SARS-CoV-2 infection induces adaptive NK cell responses by spike protein-mediated induction of HLA-E expression.

HLA-E expression plays a central role for modulation of NK cell function by interaction with inhibitory NKG2A and stimulatory NKG2C receptors on canonical and adaptive NK cells, respectively. Here, we demonstrate that infection of human primary lung tissue with SARS-CoV-2 leads to increased HLA-E expression and show that processing of the peptide YLQPRTFLL from the spike protein is primarily responsible for the strong, dose-dependent increase of HLA-E. Targeting the peptide site within the spike protein revealed that a single point mutation was sufficient to abrogate the increase in HLA-E expression. Spike-mediated induction of HLA-E differentially affected NK cell function: whereas degranulation, IFN-γ production, and target cell cytotoxicity were enhanced in NKG2C+ adaptive NK cells, effector functions were inhibited in NKG2A+ canonical NK cells. Analysis of a cohort of COVID-19 patients in the acute phase of infection revealed that adaptive NK cells were induced irrespective of the HCMV status, challenging the paradigm that adaptive NK cells are only generated during HCMV infection. During the first week of hospitalization, patients exhibited a selective increase of early NKG2C+CD57- adaptive NK cells whereas mature NKG2C+CD57+ cells remained unchanged. Further analysis of recovered patients suggested that the adaptive NK cell response is primarily driven by a wave of early adaptive NK cells during acute infection that wanes once the infection is cleared. Together, this study suggests that NK cell responses to SARS-CoV-2 infection are majorly influenced by the balance between canonical and adaptive NK cells via the HLA-E/NKG2A/C axis.

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.

Synthesis and Binding Mode Predictions of Novel Siglec-7 Ligands.

Siglec-7 regulates immune cell activity and is a promising target for immunomodulation. Here, we report the discovery of novel sialic acid derivatives binding to Siglec-7. Synthesis and affinity measurements are complemented by high-quality models of sialoside-Siglec-7 complexes based on molecular dynamics (MD) simulations on the microsecond time scale. We provide details for the predicted binding modes for the new ligands, e.g., that an extension of the carbon backbone leads to a different molecular interaction pattern with the receptor and the nearby water structure than found for known Siglec-7 ligands. Further on, we uncover some shortcomings of the GLYCAM06 and GAFF2 force fields when used for the simulation of sialoside-based glycomimetics. Our results open new opportunities for the rational design of Siglec-7 inhibitors. In addition, we provide strategies on how to use and visualize MD simulations to describe and investigate sialoside-Siglec complexes in general.

From Immunosenescence to Aging Types-Establishing Reference Intervals for Immune Age Biomarkers by Centile Estimation.

Immunological aging type definition requires establishing reference intervals from the distribution of immunosenescence biomarkers conditional on age. For 1605 individuals (18-97 years), we determined the comprehensive immune age index IMMAX from flow-cytometry-based blood cell sub-populations and identified age-specific centiles by fitting generalized additive models for location, scale, and shape. The centiles were uncorrelated with age and facilitated the categorization of individuals as immunologically slow or fast aging types. Using its 50th percentile as a reference, we rescaled the IMMAX to equivalent years of life (EYOL) and computed the immunological age gap as the difference between EYOL and chronological age. Applied to preliminary baseline and follow-up measurements from 53 participants of the Dortmund Vital Study (Clinical-Trials.gov Identifier: NCT05155397), the averaged changes in the IMMAX and EYOL conformed to the 5-year follow-up period, whereas no significant changes occurred concerning IMMAX centiles and age gap. This suggested that the participants immunologically adapted to aging and kept their relative positions within the cohort. Sex was non-significant. Methodical comparisons indicated that future confirmatory analyses with the completed follow-up examinations could rely on percentile curves estimated by simple linear quantile regression, while the selection of the immunosenescence biomarker will greatly influence the outcome, with IMMAX representing the preferable choice.

Early expansion of activated adaptive but also exhausted NK cells during acute severe SARS-CoV-2 infection.

The analysis of immunological parameters during the course of a SARS-CoV-2 infection is of great importance, both to identify diagnostic markers for the risk of a severe course of COVID-19, and to better understand the role of the immune system during the infection. By using multicolor flow cytometry we compared the phenotype of Natural Killer (NK) cells from hospitalized COVID-19 patients during early SARS-CoV-2 infection with samples from recovered and SARS-CoV-2 naïve subjects. Unsupervised high-dimensional analysis of 28-color flow cytometric data revealed a strong enrichment of NKG2C expressing NK cells in response to the acute viral infection. In addition, we found an overrepresentation of highly activated NK cell subsets with an exhausted phenotype. Moreover, our data show long-lasting phenotypic changes within the NK cell compartment that did not completely reverse up to 2 months after recovery. This demonstrates that NK cells are involved in the early innate immune response against SARS-CoV-2.

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

Analyzing the activity of the proteases granzyme B and caspase-8 inside living cells using fluorescence localization reporters.

Natural Killer (NK) cells are innate lymphocytes that are important for early immune reactions against viral infections and cancer. Their cytotoxic activity is mediated by the release of perforin and granzymes or by engaging death receptors on the surface of their target cells. Here we provide a protocol for the use of fluorescence localization reporters to measure the activity of granzyme B or caspase-8 activity inside living target cells. This method can be used to investigate how these two killing pathways are used by NK cells. By modifying the modular structure of the reporters, they can be adapted to study other cytotoxic effector cells or signaling pathways, where proteases play an important role.

A Systematic Analysis of Biological, Sociodemographic, Psychosocial, and Lifestyle Factors Contributing to Work Ability Across the Working Life Span: Cross-sectional Study.

BACKGROUND: As employees age, their physical and mental abilities decline and work ability decreases, enhancing the risk for long-term sick leave or even premature retirement. However, the relative impact of biological and environmental determinants on work ability with increasing age is poorly understood in terms of their complexity. OBJECTIVE: Previous research has shown relationships between work ability and job and individual resources, as well as specific demographic and lifestyle-related variables. However, other potentially important predictors of work ability remain unexplored, such as personality traits and biological determinants, including cardiovascular, metabolic, immunological, and cognitive abilities or psychosocial factors. Our aim was to systematically evaluate a wide range of factors to extract the most crucial predictors of low and high work ability across the working life span. METHODS: As part of the Dortmund Vital Study, 494 participants from different occupational sectors, aged between 20 and 69 years, completed the Work Ability Index (WAI) assessing employee's mental and physical resources. A total of 30 sociodemographic variables were grouped into 4 categories (social relationships, nutrition and stimulants, education and lifestyle, and work related), and 80 biological and environmental variables were grouped into 8 domains (anthropometric, cardiovascular, metabolic, immunologic, personality, cognitive, stress related, and quality of life) and have been related to the WAI. RESULTS: Using the analyses, we extracted important sociodemographic factors influencing work ability, such as education, social activities, or sleep quality, and identified age-dependent and age-independent determinants of work ability. Regression models explained up to 52% of the WAI variance. Negative predictors of work ability were chronological and immunological age, immunological inefficiency, BMI, neuroticism, psychosocial stress, emotional exhaustion, demands from work, daily cognitive failures, subclinical depression, and burnout symptoms. Positive predictors were maximum heart rate during ergometry, normal blood pressure, hemoglobin and monocyte concentration, weekly physical activity, commitment to the company, pressure to succeed, and good quality of life. CONCLUSIONS: The identified biological and environmental risk factors allowed us to evaluate work ability in its complexity. Policy makers, employers, and occupational safety and health personnel should consider the modifiable risk factors we identified to promote healthy aging at work through focused physical, dietary, cognitive, and stress-reduced preventive programs, in addition to well-balanced working conditions. This may also increase the quality of life, commitment to the job, and motivation to succeed, which are important factors to maintain or even enhance work ability in the aging workforce and to prevent early retirement. TRIAL REGISTRATION: ClinicalTrials.gov NCT05155397; https://clinicaltrials.gov/ct2/show/NCT05155397. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/32352.

The Employment of the Surface Plasmon Resonance (SPR) Microscopy Sensor for the Detection of Individual Extracellular Vesicles and Non-Biological Nanoparticles.

A wide-field surface plasmon resonance (SPR) microscopy sensor employs the surface plasmon resonance phenomenon to detect individual biological and non-biological nanoparticles. This sensor enables the detection, sizing, and quantification of biological nanoparticles (bioNPs), such as extracellular vesicles (EVs), viruses, and virus-like particles. The selectivity of bioNP detection does not require biological particle labeling, and it is achieved via the functionalization of the gold sensor surface by target-bioNP-specific antibodies. In the current work, we demonstrate the ability of SPR microscopy sensors to detect, simultaneously, silica NPs that differ by four times in size. Employed silica particles are close in their refractive index to bioNPs. The literature reports the ability of SPR microscopy sensors to detect the binding of lymphocytes (around 10 µm objects) to the sensor surface. Taken together, our findings and the results reported in the literature indicate the power of SPR microscopy sensors to detect bioNPs that differ by at least two orders in size. Modifications of the optical sensor scheme, such as mounting a concave lens, help to achieve homogeneous illumination of a gold sensor chip surface. In the current work, we also characterize the improved magnification factor of the modified SPR instrument. We evaluate the effectiveness of the modified and the primary version of the SPR microscopy sensors in detecting EVs isolated via different approaches. In addition, we demonstrate the possibility of employing translation and rotation stepper motors for precise adjustments of the positions of sensor optical elements-prism and objective-in the primary version of the SPR microscopy sensor instrument, and we present an algorithm to establish effective sensor-actuator coupling.

Impact of SARS-CoV-2 vaccination on systemic immune responses in people living with HIV.

Despite the development of vaccines, which protect healthy people from severe and life-threatening Covid-19, the immunological responses of people with secondary immunodeficiencies to these vaccines remain incompletely understood. Here, we investigated the humoral and cellular immune responses elicited by mRNA-based SARS-CoV-2 vaccines in a cohort of people living with HIV (PLWH) receiving anti-retroviral therapy. While antibody responses in PLWH increased progressively after each vaccination, they were significantly reduced compared to the HIV-negative control group. This was particularly noteworthy for the Delta and Omicron variants. In contrast, CD4+ Th cell responses exhibited a vaccination-dependent increase, which was comparable in both groups. Interestingly, CD4+ T cell activation negatively correlated with the CD4 to CD8 ratio, indicating that low CD4+ T cell numbers do not necessarily interfere with cellular immune responses. Our data demonstrate that despite the lower CD4+ T cell counts SARS-CoV-2 vaccination results in potent cellular immune responses in PLWH. However, the reduced humoral response also provides strong evidence to consider PLWH as vulnerable group and suggests subsequent vaccinations being required to enhance their protection against COVID-19.

Calibrating a Comprehensive Immune Age Metric to Analyze the Cross Sectional Age-Related Decline in Cardiorespiratory Fitness.

Cardiorespiratory fitness (CRF) is essential for sustained work ability in good health, but declines with aging, as does the functionality of the immune system, the latter process commonly referred to as immunosenescence. This study aimed to compare the capacity of immunosenescence biomarkers with chronological age for predicting low CRF in a cross-sectional sample recruited from the regional working population. CRF was determined by submaximal bicycle ergometer testing in a cross-sectional sample of 597 volunteers aged 20-70 years from the 'Dortmund Vital Study' (DVS, ClinicalTrials.gov Identifier: NCT05155397). Low CRF was scored if the ergometer test was not completed due to medical reasons or if the power output projected to a heart rate of 130 bpm divided by body mass was below sex-specific reference values of 1.25 W/kg for females and 1.5 W/kg for males, respectively. In addition to established biomarkers of immunosenescence, we calibrated a comprehensive metric of immune age to our data and compared its predictive capacity for low CRF to chronological age, while adjusting our analysis for the influence of sex, obesity, and the level of regular physical activity, by applying univariate and multiple logistic regression. While obesity, low physical activity, chronological and immune age were all associated with increased probability for low CRF in univariate analyses, multiple logistic regression revealed that obesity and physical activity together with immune age, but not chronological age, were statistically significant predictors of low CRF outcome. Sex was non-significant due to the applied sex-specific reference values. These results demonstrate that biological age assessed by our immunological metric can outperform chronological age as a predictor for CRF and indicate a potential role for immunosenescence in explaining the inter-individual variability of the age-related decline in cardiorespiratory fitness.

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.

Impact of N6-methyladenosine (m6A) modification on immunity.

N6-methyl-adenosine (m6A) is the most prevalent modification on mRNAs and long noncoding RNAs (lnRNAs) in higher eukaryotes. Modulation of m6A relies on m6A writers, erasers and readers. m6A modification contributes to diverse fundamental biological functions at the molecular, cellular, and physiological levels. The dysregulation of m6A modification has been implicated in various human diseases. Thus, m6A modification has now become a research hotspot for its potential therapeutic applications in the treatment of various cancers and diseases. The immune system is essential to provide defense against infections and cancers. This review summarizes the current knowledge about the roles of m6A in regulating immune cell functions and immune responses. Video abstract.

Immune response in ofatumumab treated multiple sclerosis patients after SARS-CoV-2 vaccination.

Objective: The pandemic induced by SARS-CoV-2 has huge implications for patients with immunosuppression that is caused by disorders or specific treatments. Especially approaches targeting B cells via anti-CD20 therapy are associated with impaired humoral immune response but sustained cellular immunity. Ofatumumab is a human anti-CD20 directed antibody applied in low dosages subcutaneously, recently licensed for Multiple Sclerosis (MS). Effects of early ofatumumab treatment on alterations of immune cell composition and immune response towards SARS-CoV-2 are incompletely understood. Methods: We here investigated immune cell alterations in early ofatumumab (Ofa) treated patients and effects on humoral (titer, neutralization capacity against wild type, Delta and Omicron) and cellular immune responses in Ofa treated MS patients following a third vaccination against SARS-CoV-2 compared to healthy controls. Results: We show that a mean treatment duration of three months in the Ofa group led to near complete B cell depletion in line with altered composition of certain CD4+ T cell subpopulations such as enhanced frequencies of naive and a decrease of non-suppressive regulatory T cells (Tregs). Titer and neutralization capacity against SARS-CoV-2 variants was impaired while cellular immune response was sustained, characterized by a strong T helper 1 profile (Th1). Interpretation: In summary, low dosage ofatumumab treatment elicits sustained depletion of B cells in line with alterations of immune cells, mainly Tregs. This is associated with impaired humoral immune response towards SARS-CoV-2 vaccination but preserved, Th1 driven cellular immunity adding crucial information regarding early effects of low dosage anti-CD20 therapy on humoral and cellular immunity.

Distinct distribution patterns of exercise-induced natural killer cell mobilization into the circulation and tumor tissue of patients with prostate cancer.

The mobilization and activation of natural killer (NK) cells have been proposed as key mechanisms promoting anti-oncogenic effects of physical exercise. Although mouse models have proven that physical exercise recruits NK cells to tumor tissue and inhibits tumor growth, this preclinical finding has not been transferred to the clinical setting yet. In this first-in-human study, we found that physical exercise mobilizes and redistributes NK cells, especially those with a cytotoxic phenotype, in line with preclinical models. However, physical exercise did not increase NK cell tumor infiltrates. Future studies should carefully distinguish between acute and chronic exercise modalities and should be encouraged to investigate more immune-responsive tumor entities.

Human NK cells responses are enhanced by CD56 engagement.

Natural Killer (NK) cells are important innate lymphocytes for effective immune responses against intracellular pathogens and tumors. CD56 is a well-known marker for human NK cells, but there is very limited information about a functional role of this surface receptor. Here, we show that engagement of CD56 can induce NK cell activation resulting in degranulation, IFN-γ secretion and morphological changes, making CD56 a potential co-activating receptor in NK cells. Interestingly, this effect was only observed in cytokine pre-activated and not in freshly isolated human NK cells, demonstrating that NK cell reactivity upon CD56 engagement was dependent on cytokine stimulation. Inhibition of Syk, PI3K, Erk, and src-family-kinases impaired CD56-mediated NK cell stimulation. Finally, we used CRISPR/Cas9 to delete CD56 from primary human NK cells. While this abolished the stimulatory effect of CD56 on pre-activated NK cells, the cytotoxic activity of NK cells against several tumor target cells was not affected by the absence of CD56. This demonstrates that the stimulating effect of CD56 on pre-activated NK cells does not have a major impact on their cytotoxic activity, but it may contribute to the function of CD56 as a fungal recognition receptor and in the NK cell developmental synapse.