Restriction of Glycolysis Increases Serial Killing Capacity of Natural Killer Cells.

Tumor cells rely heavily on glycolysis to meet their high metabolic demands. While this results in nutrient deprivation within the tumor microenvironment and has negative effects on infiltrating immune cells such as natural killer (NK) cells, it also creates a potential target for cancer therapies. Here we use Glupin, an inhibitor of glucose transporters, to study the effect of limited glucose uptake on NK cells and their anti-tumor functions. Glupin treatment effectively inhibited glucose uptake and restricted glycolysis in NK cells. However, acute treatment had no negative effect on NK cell cytotoxicity or cytokine production. Long-term restriction of glucose uptake via Glupin treatment only delayed NK cell proliferation, as they could switch to glutaminolysis as an alternative energy source. While IFN-γ production was partially impaired, long-term Glupin treatment had no negative effect on degranulation. Interestingly, the serial killing activity of NK cells was even slightly enhanced, possibly due to changes in NAD metabolism. This demonstrates that NK cell cytotoxicity is remarkably robust and insensitive to metabolic disturbances, which makes cellular metabolism an attractive target for immune-mediated tumor therapies.

A spiculated mass target model for clinical image quality control in digital mammography.

OBJECTIVES: Quality assurance of breast imaging has a long history of using test objects to optimize and follow up imaging devices. In particular, the evaluation of new techniques benefits from suitable test objects. The applicability of a phantom consisting of spiculated masses to assess image quality and its dependence on dose in flat field digital mammography (FFDM) and digital breast tomosynthesis systems (DBT) is investigated. METHODS: Two spiculated masses in five different sizes each were created from a database of clinical tumour models. The masses were produced using 3D printing and embedded into a cuboid phantom. Image quality is determined by the number of spicules identified by human observers. RESULTS: The results suggest that the effect of dose on spicule detection is limited especially in cases with smaller objects and probably hidden by the inter-reader variability. Here, an average relative inter-reader variation of the counted number of 31% was found (maximum 83%). The mean relative intra-reader variability was found to be 17%. In DBT, sufficiently good results were obtained only for the largest masses. CONCLUSIONS: It is possible to integrate spiculated masses into a cuboid phantom. It is easy to print and should allow a direct and prompt evaluation of the quality status of the device by counting visible spicules. Human readout presented the major uncertainty in this study, indicating that automated readout may improve the reproducibility and consistency of the results considerably. ADVANCES IN KNOWLEDGE: A cuboid phantom including clinical objects as spiculated lesion models for visual assessing the image quality in FFDM and DBT was developed and is introduced in this work. The evaluation of image quality works best with the two larger masses with 21 spicules.

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection.

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future.

Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitor Improves Leukocyte Energy Metabolism in Hereditary Hemorrhagic Telangiectasia.

The interplay between hypoxia-inducible factors (HIFs) and transforming growth factor beta (TGF-ß) is critical for both inflammation and angiogenesis. In hereditary hemorrhagic telangiectasia (HHT), we have previously observed that impairment of the TGF-ß pathway is associated with downregulation of HIF-1α. HIF-1α accumulation is mandatory in situations of altered energy demand, such as during infection or hypoxia, by adjusting cell metabolism. Leukocytes undergo a HIF-1α-dependent switch from aerobic mitochondrial respiration to anaerobic glycolysis (glycolytic switch) after stimulation and during differentiation. We postulate that the decreased HIF-1α accumulation in HHT leads to a clinically observed immunodeficiency in these patients. Examination of HIF-1α and its target genes in freshly isolated peripheral blood mononuclear cells (PBMCs) from HHT patients revealed decreased gene expression and protein levels of HIF-1α and HIF-1α-regulated glycolytic enzymes. Treatment of these cells with the HIF-prolyl hydroxylase inhibitor, Roxadustat, rescued their ability to accumulate HIF-1α protein. Functional analysis of metabolic flux using a Seahorse FX extracellular flux analyzer showed that the extracellular acidification rate (indicator of glycolytic turnover) after Roxadustat treatment was comparable to non-HHT controls, while oxygen consumption (indicator of mitochondrial respiration) was slightly reduced. HIF stabilization may be a potential therapeutic target in HHT patients suffering from infections.

Technical note: Realization and uncertainty analysis for an adjustable 3D structured breast phantom in digital breast tomosynthesis.

BACKGROUND: Projection imaging phantoms are often optimized for 2-dimensional image characteristics in homogeneous backgrounds. Therefore, evaluation of image quality in tomosynthesis (DBT) lacks accepted and established phantoms. PURPOSE: We describe a 3D breast phantom with a structured, variable background. The phantom is an adaptable and advanced version of the L1 phantom by Cockmartin et al. Phantom design and its use for quality assurance measurements for DBT devices are described. Four phantoms were compared to assess the objectivity. METHODS: The container size was increased to a diameter of 24 cm and a total height of 53.5 mm. Spiculated masses were replaced by five additional non-spiculated masses for higher granularity in threshold diameter resolution. These patterns are adjustable to the imaging device. The masses were printed in one session with a base layer using two-component 3D printing. New materials compared to the L1 phantom improved the attenuation difference between the lesion models and the background. Four phantoms were built and intra-human observer, inter-human observer and inter-phantom variations were determined. The latter assess the reproducibility of the phantom production. Coefficients of variance (V) were calculated for all three variations. RESULTS: The difference of the attenuation coefficients between the lesion models and the background was 0.20 cm-1 (with W/Al at 32 kV, equivalent to 19-20 keV effective energy) compared to 0.21 cm-1 for 50/50 glandular/adipose breast tissue and cancerous lesions. PMMA equivalent thickness of the phantom was 47.0 mm for the Siemens Mammomat Revelation. For the masses, the V i n t r a $V_{intra}$ for the intra-observer variation was 0.248, the averaged inter-observer variation, V ¯ i n t e r $\overline{V}_{inter}$ was 0.383. V p h a n t o m $V_{phantom}$ for phantom variance was 0.321. For the micro-calcifications, V i n t r a $V_{intra}$ was 0.0429, V ¯ i n t e r = $\overline{V}_{inter}=$ 0.0731 and V p h a n t o m = $V_{phantom}=$ 0.0759. CONCLUSIONS: Position, orientation and shape of the masses are reproducible and attenuation differences appropriate. The phantom presented proved to be a candidate test object for quality control.

Superior antiviral activity of IFNß in genital HSV-1 infection.

Type I interferons (IFNs) present the first line of defense against viral infections, providing antiviral, immunomodulatory and antiproliferative effects. The type I IFN family contains 12 IFNα subtypes and IFNß, and although they share the same receptor, they are classified as non-redundant, capable to induce a variety of different IFN-stimulated genes. However, the biological impact of individual subtypes remains controversial. Recent data propose a subtype-specificity of type I IFNs revealing unique effector functions for different viruses and thus expanding the implications for IFNα-based antiviral immunotherapies. Despite extensive research, drug-resistant infections with herpes simplex virus type 1 (HSV-1), which is the common agent of recurrent orogenital lesions, are still lacking a protective or curing therapeutic. However, due to the risk of generalized infections in immunocompromised hosts as well as the increasing incidence of resistance to conventional antiherpetic agents, HSV infections raise major health concerns. Based on their pleiotropic effector functions, the application of type I IFNs represents a promising approach to inhibit HSV-1 replication, to improve host immunity and to further elucidate their qualitative differences. Here, selective IFNα subtypes and IFNß were evaluated for their therapeutic potential in genital HSV-1 infections. Respective in vivo studies in mice revealed subtype-specific differences in the reduction of local viral loads. IFNß had the strongest antiviral efficacy against genital HSV-1 infection in mice, whereas IFNα1, IFNα4, and IFNα11 had no impact on viral loads. Based on flow cytometric analyses of underlying immune responses at local and peripheral sites, these differences could be further assigned to specific modulations of the antiviral immunity early during HSV-1 infection. IFNß led to enhanced systemic cytokine secretion and elevated cytotoxic responses, which negatively correlated with viral loads in the vaginal tract. These data provide further insights into the diversity of type I IFN effector functions and their impact on the immunological control of HSV-1 infections.

Inhibition of Glucose Uptake Blocks Proliferation but Not Cytotoxic Activity of NK Cells.

Tumor cells often have very high energy demands. Inhibition of glucose uptake is therefore a possible approach to limit the proliferation and survival of transformed cells. However, immune cells also require energy to initiate and to maintain anti-tumor immune reactions. Here, we investigate the effect of Glutor, an inhibitor of glucose transporters, on the function of human Natural Killer (NK) cells, which are important for the immunosurveillance of cancer. Glutor treatment effectively inhibits glycolysis in NK cells. However, acute treatment with the inhibitor has no effect on NK cell effector functions. Prolonged inhibition of glucose uptake by Glutor prevents the proliferation of NK cells, increases their pro-inflammatory regulatory function and reduces the stimulation-dependent production of IFN-γ. Interestingly, even after prolonged Glutor treatment NK cell cytotoxicity and serial killing activity were still intact, demonstrating that cytotoxic NK cell effector functions are remarkably robust against metabolic disturbances.

A multi-modality physical phantom for mimicking tumor heterogeneity patterns in PET/CT and PET/MRI.

BACKGROUND: Hybrid imaging (e.g., positron emission tomography [PET]/computed tomography [CT], PET/magnetic resonance imaging [MRI]) helps one to visualize and quantify morphological and physiological tumor characteristics in a single study. The noninvasive characterization of tumor heterogeneity is essential for grading, treatment planning, and following-up oncological patients. However, conventional (CONV) image-based parameters, such as tumor diameter, tumor volume, and radiotracer activity uptake, are insufficient to describe tumor heterogeneities. Here, radiomics shows promise for a better characterization of tumors. Nevertheless, the validation of such methods demands imaging objects capable of reflecting heterogeneities in multi-modality imaging. We propose a phantom to simulate tumor heterogeneity repeatably in PET, CT, and MRI. METHODS: The phantom consists of three 50-ml plastic tubes filled partially with acrylic spheres of S1: 1.6 mm, S2: 50%(1.6 mm)/50%(6.3 mm), or S3: 6.3-mm diameter. The spheres were fixed to the bottom of each tube by a plastic grid, yielding one sphere free homogeneous region and one heterogeneous (S1, S2, or S3) region per tube. A 3-tube phantom and its replica were filled with a fluorodeoxyglucose (18F) solution for test-retest measurements in a PET/CT Siemens TPTV and a PET/MR Siemens Biograph mMR system. A number of 42 radiomic features (10 first order and 32 texture features) were calculated for each phantom region and imaging modality. Radiomic features stability was evaluated through coefficients of variation (COV) across phantoms and scans for PET, CT, and MRI. Further, the Wilcoxon test was used to assess the capability of stable features to discriminate the simulated phantom regions. RESULTS: The different patterns (S1-S3) did present visible heterogeneity in all imaging modalities. However, only for CT and MRI, a clear visual difference was present between the different patterns. Across all phantom regions in PET, CT, and MR images, 10, 16, and 21 features out of 42 evaluated features in total had a COV of 10% or less. In particular, CONV, histogram, and gray-level run length matrix features showed high repeatability for all the phantom regions and imaging modalities. Several of repeatable texture features allowed the image-based discrimination of the different phantom regions (p < 0.05). However, depending on the feature, different pattern discrimination capabilities were found for the different imaging modalities. CONCLUSION: The proposed phantom appears suitable for simulating heterogeneities in PET, CT, and MRI. We demonstrate that it is possible to select radiomic features for the readout of the phantom. Most of these features had been shown to be relevant in previous clinical studies.

Simultaneous or Prior Activation of Intrahepatic Type I Interferon Signaling Leads to Hepatitis B Virus Persistence in a Mouse Model.

It remains controversial how interferon (IFN) response contributes to hepatitis B virus (HBV) control and pathogenesis. A previous study identified that hydrodynamic injection (HI) of type I IFN (IFN-I) inducer polyinosinic-poly(C) [poly(I·C)] leads to HBV clearance in a chronic HBV mouse model. However, recent studies have suggested that premature IFN-I activation in the liver may facilitate HBV persistence. In the present study, we investigated how the early IFN-I response induces an immunosuppressive signaling cascade and thus causes HBV persistence. We performed HI of the plasmid adeno-associated virus (pAAV)/HBV1.2 into adult BALB/c mice to establish an adult acute HBV replication model. Activation of the IFN-I signaling pathway following poly(I·C) stimulation or murine cytomegalovirus (MCMV) infection resulted in subsequent HBV persistence. HI of poly(I·C) with the pAAV/HBV1.2 plasmid resulted in not only the production of IFN-I and the anti-inflammatory cytokine interleukin-10 (IL-10) but also the expansion of intrahepatic regulatory T cells (Tregs), Kupffer cells (KCs), and myeloid-derived suppressor cells (MDSCs), all of which impaired the T cell response. However, when poly(I·C) was injected at day 14 after the HBV plasmid injection, it significantly enhanced HBV-specific T cell responses. In addition, interferon-alpha/beta receptor (IFNAR) blockade rescued T cell response by downregulating IL-10 expression and decreasing Treg and KC expansion. Consistently, Treg depletion or IL-10 blockade also controlled HBV replication. IMPORTANCE IFN-I plays a double-edged sword role during chronic HBV infection. Here, we identified that application of IFN-I at different time points causes contrast outcomes. Activation of the IFN-I pathway before HBV replication induces an immunosuppressive signaling cascade in the liver and consequently caused HBV persistence, while IFN-I activation post HBV infection enhances HBV-specific T cell responses and thus promotes HBV clearance. This result provided an important clue to the mechanism of HBV persistence in adult individuals.

Metabolic requirements of NK cells during the acute response against retroviral infection.

Natural killer (NK) cells are important early responders against viral infections. Changes in metabolism are crucial to fuel NK cell responses, and altered metabolism is linked to NK cell dysfunction in obesity and cancer. However, very little is known about the metabolic requirements of NK cells during acute retroviral infection and their importance for antiviral immunity. Here, using the Friend retrovirus mouse model, we show that following infection NK cells increase nutrient uptake, including amino acids and iron, and reprogram their metabolic machinery by increasing glycolysis and mitochondrial metabolism. Specific deletion of the amino acid transporter Slc7a5 has only discrete effects on NK cells, but iron deficiency profoundly impaires NK cell antiviral functions, leading to increased viral loads. Our study thus shows the requirement of nutrients and metabolism for the antiviral activity of NK cells, and has important implications for viral infections associated with altered iron levels such as HIV and SARS-CoV-2.

Performance of semiconductor dosimeters with a range of radiation qualities used for mammography: A calibration laboratory study.

PURPOSE: To investigate the radiation quality dependence of the response of commercial semiconductor-based dosimeters, and to estimate potential errors and uncertainties related to different measurement and calibration scenarios. METHODS: All measurement results were compared to reference values measured at the IAEA dosimetry laboratory which is traceable to the international system of units (SI). Energy dependence of the response of eight semiconductor dosimeters were determined for five different anode-filter combinations and tube voltages from 25 to 35 kV. For systems capable of deriving half value layer (HVL) and tube voltage from measurements, calibration coefficients for these measurements were calculated. RESULTS: For six dosimeters, the maximum deviations from the reference value of the air kerma measurement were within ±5% as required by IEC 61674. Calibration coefficients for radiation qualities (anode-filter and tube voltage combinations) relative to reference radiation quality Mo-Mo 28 kV deviate up to 12%. HVL and tube voltage measurements exhibited deviations up to 11% and 10%, respectively. CONCLUSIONS: The air kerma responses of modern semiconductor dosimeters have a small energy dependence. However, no dosimeter tested complied with the accuracy limits stated by the manufacturer for tube voltage measurements, and only two dosimeters complied with the limits for HVL measurements. Absolute measurement of HVL and tube voltage with semiconductor dosimeters have to be verified for actual clinical radiation conditions on clinical mammography systems. Semiconductor dosimeters can be used for quality control measurements if individual calibration coefficients are available for the radiation condition applied. If other conditions are applied, additional uncertainty needs to be considered, particularly in the case of HVL and tube voltage measurements.

Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses.

Natural Killer (NK) cells are lymphocytes with an important role in anti-tumour responses. NK cells bridge the innate and adaptive arms of the immune system; they are primed for immediate anti-tumour function but can also have prolonged actions alongside the adaptive T cell response. However, the key signals and cellular processes that are required for extended NK cell responses are not fully known. Herein we show that murine NK cell interaction with tumour cells induces the expression of CD25, the high affinity IL2 receptor, rendering these NK cells highly sensitive to the T cell-derived cytokine IL2. In response to IL2, CD25high NK cells show robust increases in metabolic signalling pathways (mTORC1, cMyc), nutrient transporter expression (CD71, CD98), cellular growth and in NK cell effector functions (IFNγ, granzyme B). Specific ligation of an individual activating NK cell receptor, NK1.1, showed similar increases in CD25 expression and IL2-induced responses. NK cell receptor ligation and IL2 collaborate to induce mTORC1/cMyc signalling leading to high rates of glycolysis and oxidative phosphorylation (OXPHOS) and prolonged NK cell survival. Disrupting mTORC1 and cMyc signalling in CD25high tumour interacting NK cells prevents IL2-induced cell growth and function and compromises NK cell viability. This study reveals that tumour cell interactions and T cell-derived IL2 cooperate to promote robust and prolonged NK cell anti-tumour metabolic responses.

IFI16 Targets the Transcription Factor Sp1 to Suppress HIV-1 Transcription and Latency Reactivation.

The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently of immune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Highly prevalent clade C HIV-1 strains are more resistant to IFI16 and less dependent on Sp1 than other HIV-1 subtypes. Furthermore, inhibition of Sp1 by IFI16 or pharmacologically by Mithramycin A suppresses reactivation of latent HIV-1 in CD4+ T cells. Finally, IFI16 also inhibits retrotransposition of LINE-1, known to engage Sp1, and murine IFI16 homologs restrict Friend retrovirus replication in mice. Thus, IFI16 restricts retroviruses and retrotransposons by interfering with Sp1-dependent gene expression, and evasion from this restriction may facilitate spread of HIV-1 subtype C.

Comparison of a personalized breast dosimetry method with standard dosimetry protocols.

Average glandular dose (AGD) in digital mammography crucially depends on the estimation of breast glandularity. In this study we compared three different methods of estimating glandularities according to Wu, Dance and Volpara with respect to resulting AGDs. Exposure data from 3050 patient images, acquired with a GE Senographe Essential constituted the study population of this work. We compared AGD (1) according to Dance et al. applying custom g, c, and s factors using HVL, breast thickness, patient age and incident air kerma (IAK) from the DICOM headers; (2) according to Wu et al. as determined by the GE system; and (3) AGD derived with the Dance model with personalized c factors using glandularity determined with the Volpara (Volpara Solutions, Wellington, New Zealand) software (Volpare AGD). The ratios of the resulting AGDs were analysed versus parameters influencing dose. The highest deviation between the resulting AGDs was found in the ratio of GE AGD to Volpara AGD for breast thicknesses between 20 and 40 mm (ratio: 0.80). For thicker breasts this ratio is close to one (1 ± 0.02 for breast thicknesses >60 mm). The Dance to Volpara ratio was between 0.86 (breast thickness 20-40 mm) and 0.99 (>80 mm), and Dance/GE AGD was between 1.07 (breast thickness 20-40 mm) and 0.98 (41-60, and >80 mm). Glandularities by Volpara were generally smaller than the one calculated with the Dance method. This effect is most pronounced for small breast thickness and older ages. Taking the considerable divergences between the AGDs from different methods into account, the selection of the method should by done carefully. As the Volpara method provides an analysis of the individual breast tissue, while the Wu and the Dance methods use look up tables and custom parameter sets, the Volpara method might be more appropriate if individual ADG values are sought. For regulatory purposes and comparison with diagnostic reference values, the method to be used needs to be defined exactly and clearly be stated. However, it should be accepted that dose values calculated with standardized models, like AGD and also effective dose, are afflicted with a considerable uncertainty budgets that need to be accounted for in the interpretation of these values.

Effects of Friend Virus Infection and Regulatory T Cells on the Antigen Presentation Function of B Cells.

Friend virus (FV) is a naturally occurring mouse retrovirus that infects dividing cells of the hematopoietic lineage, including antigen-presenting cells (APCs). The infection of APCs by viruses often induces their dysfunction, and it has been shown that FV infection reduces the ability of dendritic cells (DCs) to prime critical CD8+ T cell responses. Nonetheless, mice mount vigorous CD8+ T cell responses, so we investigated whether B cells might serve as alternative APCs during FV infection. Direct ex vivo analysis of B cells from FV-infected mice revealed that infected but not uninfected B cells upregulated expression of the costimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC-II) molecules. Furthermore, in vitro studies showed that, compared to uninfected B cells from the same mice, the FV-infected B cells had significantly enhanced APC function, as measured by their capacity to prime CD8+ T cell activation and proliferation. Thus, in contrast to DCs, infection of B cells with FV enhanced their APC capacity and ability to stimulate the CD8+ T cell responses essential for virus control. FV infections also induce the activation and expansion of regulatory T cells (Tregs), so it was of interest to determine the impact of Tregs on B cell activation. The upregulation of costimulatory molecule expression and APC function of B cells was even more strongly enhanced by in vivo depletion of regulatory T cells than infection. Thus, Tregs exert potent homeostatic suppression of B cell activation that is partially overcome by FV infection.IMPORTANCE The primary role of B cells in immunity is considered the production of pathogen-specific antibodies, but another, less-well-studied, function of B cells is to present foreign antigens to T cells to stimulate their activation and proliferation. Dendritic cells (DCs) are considered the most important antigen-presenting cells (APCs) for CD8+ T cells, but DCs lose APC function when infected with Friend virus (FV), a model retrovirus of mice. Interestingly, B cells were better able to stimulate CD8+ T cell responses when they were infected with FV. We also found that the activation status of B cells under homeostatic conditions was potently modulated by regulatory T cells. This study illustrates an important link between B cell and T cell responses and illustrates an additional mechanism by which regulatory T cells suppress critical T cell responses during viral infections.

Chronic retroviral infection of mice promotes tumor development, but CD137 agonist therapy restores effective tumor immune surveillance.

T cell responses are crucial for anti-tumor immunity. In chronic viral infections, anti-tumor T cell responses can be compromised due to various immunological mechanisms, including T cell exhaustion. To study mechanisms of anti-tumor immunity during a chronic viral infection, we made use of the well-established Friend virus (FV) mouse model. Chronically FV-infected mice are impaired in their ability to reject FBL-3 cells-a virus-induced tumor cell line of C57BL/6 origin. Here we aimed to explore therapeutic strategies to overcome the influence of T cell exhaustion during chronic viral infection, and reactivate effector CD8+ and CD4+ T cells to eliminate tumor cells. For T cell stimulation, agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily members CD137 and CD134 were used, because they were reported to augment the cytotoxic program of T cells. αCD137 agonistic therapy, but not αCD134 agonistic therapy, resulted in FBL-3 tumor elimination in chronically FV-infected mice. CD137 stimulation significantly enhanced the cytotoxic activity of both CD4+ and CD8+ T cells, which were both required for efficient tumor control. Our study suggests that agonistic antibodies to CD137 can efficiently enhance anti-tumor immunity even in the setting of chronic viral infection, which might have promising therapeutic applications.

Friend retrovirus infection induces the development of memory-like natural killer cells.

Traditionally, NK cells belong to the innate immune system and eliminate virus-infected cells through their germline-encoded receptors. However, NK cells were recently reported to possess memory-like functions that were predominantly provided by hepatic NK cells. Memory properties were mainly documented in contact hypersensitivity models or during cytomegalovirus infections. However, the precise role and the physiologic importance of memory-like NK cells during retroviral infections are still under investigation. Here, we show that Friend retrovirus (FV) infection of mice induced a population of phenotypically memory-like NK cells at 28 days post infection. Upon secondary antigen encounter, these NK cells showed an increased production of the pro-inflammatory cytokines IFNγ and TNFα as well as the death ligand FasL in comparison to naïve NK cells. Furthermore, we found an augmented elimination of antigen-matched but not antigen-mismatched target cells by these memory-like NK cells. In adoptive cell transfer experiments, equal antiviral activities of splenic and hepatic memory-like NK cells during the late phase of acute FV infection were found. Our results strongly imply the existence and antiviral activity of spleen and liver memory-like NK cells in FV infection, which efficiently respond upon secondary exposure to retroviral antigens.

The Cytotoxic Activity of Natural Killer Cells Is Suppressed by IL-10+ Regulatory T Cells During Acute Retroviral Infection.

Natural killer (NK) cells play a key role in host defense against cancer and viral infections. It was shown that NK cells are important for the control of acute retroviral infections, but their antiviral activity depends on multiple parameters such as viral inoculation dose, interactions with myeloid cell types and the cytokine milieu. In addition, during an ongoing retroviral infection regulatory T cells (Tregs) can suppress NK cell functions. However, the precise role of Tregs on the initial NK cell response and their immediate antiviral activity after an acute retroviral infection is still unknown. Here we show that thymus-derived Tregs suppress the proliferation, effector functions and cytotoxicity of NK cells very early during acute Friend Retrovirus (FV) infection. Tregs exhibited an activated phenotype and increased the production of the immunosuppressive cytokines IL-10 and TGF-ß after FV infection of mice. Neutralization of the immunosuppressive cytokine IL-10 resulted in a significant augmentation of NK cell functions. Although the activation of dendritic cells (DCs) and macrophages as well as the IL-15 cytokine levels were increased after Treg depletion, Tregs mainly affect the NK cell activity in an IL-10-regulated pathway. In this study we demonstrate an IL-10-dependent suppression of NK cells by activated Tregs during the first days of a retroviral infection.

Dose of Retroviral Infection Determines Induction of Antiviral NK Cell Responses.

Natural killer (NK) cells are part of the innate immune system and recognize virus-infected cells as well as tumor cells. Conflicting data about the beneficial or even detrimental role of NK cells in different infectious diseases have been described previously. While the type of pathogen strongly influences NK cell functionality, less is known about how the infection dose influences the quality of a NK cell response against retroviruses. In this study, we used the well-established Friend retrovirus (FV) mouse model to investigate the impact of virus dose on the induction of antiviral NK cell functions. High-dose virus inoculation increased initial virus replication compared to that with medium- or low-dose viral challenge and significantly improved NK cell activation. Antiviral NK cell activity, including in vivo cytotoxicity toward infected target cells, was also enhanced by high-dose virus infection. NK cell activation following high-dose viral challenge was likely mediated by activated dendritic cells (DCs) and macrophages and the NK cell-stimulating cytokines interleukin 15 (IL-15) and IL-18. Neutralization of these cytokines decreased NK cell functions and increased viral loads, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we demonstrate that virus dose positively correlates with antiviral NK cell activity and function, which are at least partly driven by IL-15 and IL-18. Our results suggest that NK cell activity may be therapeutically enhanced by administering IL-15 and IL-18 in virus infections that inadequately activate NK cells.IMPORTANCE In infections with retroviruses, like HIV and FV infection of mice, NK cells clearly mediate antiviral activities, but they are usually not sufficient to prevent severe pathology. Here we show that the initial infection dose impacts the induction of an antiviral NK cell response during an acute retroviral infection, which had not investigated before. High-dose infection resulted in a strong NK cell functionality, whereas no antiviral activities were detected after low- or medium-dose infection. Interestingly, DCs and macrophages were highly activated after high-dose FV challenge, which corresponded with increased levels of NK cell-stimulating cytokines IL-15 and IL-18. IL-15 and IL-18 neutralization decreased NK cell functions, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we show the importance of cytokines for NK cell activation in retroviral infections; our findings suggest that immunotherapy combining the well-tolerated cytokines IL-15 and IL-18 might be an interesting approach for antiretroviral treatment.

Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection.

CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with retroviruses. Similar to textbook knowledge the most important function of CD4+ T cells during an acute retrovirus infection seems to be their helper function for other immune cells. Whereas there was no direct anti-viral activity of CD4+ T cells during acute Friend Virus (FV) infection, they were absolutely required for the control of chronic infection. During chronic FV infection a population of activated FV-specific CD4+ T cells did not express cytotoxic molecules, but Fas Ligand that can induce Fas-induced apoptosis in target cells. Using an MHC II-restricted in vivo CTL assay we demonstrated that FV-specific CD4+ T cells indeed mediated cytotoxic effects against FV epitope peptide loaded targets. CD4 + CTL killing was also detected in FV-infected granzyme B knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies against FasL, which identified the Fas/FasL pathway as critical cytotoxic mechanism during chronic FV infection. Interestingly, targeting the co-stimulatory receptor CD137 with an agonistic antibody enhanced CD4+ T cell cytotoxicity. This immunotherapy may be an interesting new approach for the treatment of chronic viral infections.