Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

Role for early-differentiated natural killer cells in infectious mononucleosis.

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) immunoglobulin-like receptor(-) NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus.

Innate Immune Recognition of EBV.

The ability of Epstein-Barr virus (EBV) to establish latency despite specific immune responses and to successfully persist lifelong in the human host shows that EBV has developed powerful strategies and mechanisms to exploit, evade, abolish, or downsize otherwise effective immune responses to ensure its own survival. This chapter focuses on current knowledge on innate immune responses against EBV and its evasion strategies for own benefit and summarizes the questions that remain to be tackled. Innate immune reactions against EBV originate both from the main target cells of EBV and from nontarget cells, which are elements of the innate immune system. Thus, we structured our review accordingly but with a particular focus on the innate recognition of EBV in its two stages in its life cycle, latent state and lytic replication. Specifically, we discuss (I) innate sensing and resulting innate immune responses against EBV by its main target cells, focusing on (i) EBV transmission between epithelial cells and B cells and their life cycle stages; and (ii) elements of innate immunity in EBV's target cells. Further, we debate (II) the innate recognition and resulting innate immune responses against EBV by cells other than the main target cells, focusing on (iii) myeloid cells: dendritic cells, monocytes, macrophages, and neutrophil granulocytes; and (iv) natural killer cells. Finally, we address (III) how EBV counteracts or exploits innate immunity in its latent and lytic life cycle stages, concentrating on (v) TLRs; (vi) EBERs; and (vii) microRNAs.

A distinct subpopulation of human NK cells restricts B cell transformation by EBV.

NK cells constitute the first line of defense against pathogens and transformed cells. They mature in secondary lymphoid organs, including tonsils, where common pathogens, such as EBV, enter the host and potentially imprint differentiating cells, which then patrol the body via the blood stream. Therefore, we set out to characterize a distinct human NK cell population in tonsils that produces high amounts of the immunomodulatory and antiviral cytokine IFN-γ. We found that the tonsilar IFN-γ(high) NK cell subset is CD56(bright)NKG2A(+)CD94(+)CD54(+)CD62L(-), is present in tonsils ex vivo and is more mature than other CD56(bright) NK cells in tonsils and less mature than other NK cells in blood, shows very low plasticity even after prolonged cytokine stimulation, accumulates in tonsils of EBV carriers, and is able to potently restrict EBV-induced transformation of B cells. Thus, we characterized a distinct and stable IFN-γ(high) NK cell subpopulation that can specifically restrict malignant transformation of EBV-infected B cells. This subset should be exploited for future development of cell-based therapeutic approaches in EBV-associated malignancies.

TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells.

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.

Impaired IFN-γ production and proliferation of NK cells in multiple sclerosis.

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patients.

Human NK cells kill resting but not activated microglia via NKG2D- and NKp46-mediated recognition.

Microglia are resident macrophage-like APCs of the CNS. To avoid escalation of inflammatory processes and bystander damage within the CNS, microglia-driven inflammatory responses need to be tightly regulated and both spatially and temporally restricted. Following traumatic, infectious, and autoimmune-mediated brain injury, NK cells have been found in the CNS, but the functional significance of NK cell recruitment and their mechanisms of action during brain inflammation are not well understood. In this study, we investigated whether and by which mechanisms human NK cells might edit resting and activated human microglial cells via killing in vitro. IL-2-activated NK cells efficiently killed both resting allogeneic and autologous microglia in a cell-contact-dependent manner. Activated NK cells rapidly formed synapses with human microglial cells in which perforin had been polarized to the cellular interface. Ab-mediated NKG2D and NKp46 blockade completely prevented the killing of human microglia by activated NK cells. Up-regulation of MHC class I surface expression by TLR4 stimulation protected microglia from NK cell-mediated killing, whereas MHC class I blockade enhanced cytotoxic NK cell activity. These data suggest that brain-infiltrating NK cells might restrict innate and adaptive immune responses within the human CNS via elimination of resting microglia.

Regulatory NK-cell functions in inflammation and autoimmunity.

Natural killer (NK) cells were viewed traditionally as cytotoxic effector cells whose rapid killing of infected and transformed cells without preactivation provides a first line of defense prior to the initiation of an adaptive immune response against infection and tumor development. However, it has become clear that NK cells interact with various components of the immune system, and therefore have the potential to function as regulatory cells. While NK cells can assist in dendritic cell (DC) maturation and T-cell polarization, increasing evidence indicates that NK cells can also prevent and limit adaptive (auto) immune responses via killing of autologous myeloid and lymphoid cells. Investigating immunoregulatory NK-cell functions might generate exciting insights into the reciprocal regulation between NK-cell-mediated innate immunity and adaptive immune responses, improve our capacity to monitor these cells as surrogate markers for disease activity and treatment responses in autoimmune diseases, and, perhaps, provide new prospects for NK cell-directed therapies.

Tonsillar CD56brightNKG2A+ NK cells restrict primary Epstein-Barr virus infection in B cells via IFN-γ.

Natural killer (NK) cells constitute the first line of defense against viruses and cancers cells. Epstein-Barr virus (EBV) was the first human virus to be directly implicated in carcinogenesis, and EBV infection is associated with a broad spectrum of B cell lymphomas. How NK cells restrict EBV-associated oncogenesis is not understood. Here, we investigated the efficacies and mechanisms of distinct NK cell subsets from tonsils, the portal of entry of EBV, in limiting EBV infection in naïve, germinal center-associated and memory B cells. We found that CD56bright and NKG2A expression sufficiently characterizes the potent anti-EBV capacity of tonsillar NK cells. We observed restriction of EBV infection in B cells as early as 18 hours after infection. The restriction was most efficient in naïve B cells and germinal center-associated B cells, the B cell subsets that exhibited highest susceptibility to EBV infection in vitro. IFN-γ release by and partially NKp44 engagement of CD56bright and NKG2A positive NK cells mediated the restriction that eventually inhibited B-cell transformation. Thus, harnessing CD56brightNKG2A+ NK cell function might be promising to improve treatment strategies that target EBV-associated B cell lymphomas.