Additional use of α-IgM antibodies potentiates CpG ODN2006-induced B cell activation by targeting mainly naïve and marginal zone-like B cells.

CpG ODN2006 is widely used as a potent B cell stimulant in vitro and in vivo. However, it shows a deficit in targeting naïve B cells in vitro. In this study, we investigated whether α-IgM can support ODN2006-induced effects on B cells to obtain enhanced activation with focus on different B cell subsets. Our results delineated robust B cell activation, shown by increased activation marker expression and cytokine secretion by each agent alone, and further augmented when used in combination. Interestingly, α-IgM targeted mainly naïve and marginal zone-like B cells, thus complementing the pronounced effects of ODN2006 on memory B cells and achieving optimal activation for all B cell subsets. Taken together, combining ODN2006 and α-IgM is beneficial for in vitro activation including all B cell subsets. Furthermore, our results suggest that α-IgM could enhance efficacy of ODN2006 in vivo with further need of investigation.

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.

Neutralizing antibody responses 300 days after SARS-CoV-2 infection and induction of high antibody titers after vaccination.

Neutralizing antibodies against SARS-CoV-2 are important to protect against infection and/or disease. Using an assay to detect antibodies directed against the receptor binding domain (RBD) of SARS-CoV-2 Spike, we identified individuals with SARS-CoV-2 infection after an outbreak at a local health institution. All but one COVID-19 patient developed detectable anti-RBD antibodies and 77% had virus neutralizing antibody titers of >1:25. Antibody levels declined slightly over time. However, we still detected virus neutralizing antibody titers in 64% of the COVID-19 patients at >300 days after infection, demonstrating durability of neutralizing antibody levels after infection. Importantly, full COVID-19 vaccination of these individuals resulted in higher antibody titers compared to fully vaccinated individuals in the absence of prior infection. These data demonstrate long-lived antibody-mediated immunity after SARS-CoV-2 infection, and a clear benefit of two vaccine doses for recovered individuals.

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.

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.

SLAM family receptors in natural killer cells - Mediators of adhesion, activation and inhibition via cis and trans interactions.

SLAM family receptors are important for the fine-tuning of immune reactions. Their expression is restricted to cells of hematopoietic origin and most SLAM family receptors are their own ligand. Here we review how these receptors are involved in regulating the functions of Natural Killer (NK) cells. We discuss that promoting cellular adhesion may be a main function of SLAM family receptors in NK cells. The homophilic interactions of SLAM family receptors can not only occur in trans between different cells, but also in cis on the surface of the same cell. This cis interaction additionally modulates the function of the receptors and subsequently affects the activities of NK cells. Finally, SLAM-family receptors can also mediate inhibitory signals under certain conditions. These inhibitory signals can contribute to the functional maturation of NK cells during NK cell education. Therefore, SLAM family receptors are critically involved in many aspects of NK cell functionality.

Recruitment of activating NK-cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions.

Membrane microdomains play an important role in the regulation of natural killer (NK) cell activities. These cholesterol-rich membrane domains are enriched at the activating immunological synapse and several activating NK-cell receptors are known to localize to membrane microdomains upon receptor engagement. In contrast, inhibitory receptors do not localize in these specialized membrane domains. In addition, the functional competence of educated NK cells correlates with a confinement of activating receptors in membrane microdomains. However, the molecular basis for this confinement is unknown. Here, we investigate the structural requirements for the recruitment of the human-activating NK-cell receptors NKG2D and 2B4 to detergent-resistant membrane fractions in the murine BA/F3 cell line and in the human NK-cell line NKL. This stimulation-dependent recruitment occurred independently of the intracellular domains of the receptors. However, either interfering with the association between NKG2D and DAP10, or mutating the transmembrane region of 2B4 impacted the recruitment of the receptors to detergent-resistant membrane fractions and modulated the function of 2B4 in NK cells. Our data suggest a potential interaction between the transmembrane region of NK-cell receptors and membrane lipids as a molecular mechanism involved in determining the membrane confinement of activating NK-cell receptors.

Measuring the immune system: a comprehensive approach for the analysis of immune functions in humans.

The immune system is essential to provide protection from infections and cancer. Disturbances in immune function can therefore directly affect the health of the affected individual. Many extrinsic and intrinsic factors such as exposure to chemicals, stress, nutrition and age have been reported to influence the immune system. These influences can affect various components of the immune system, and we are just beginning to understand the causalities of these changes. To investigate such disturbances, it is therefore essential to analyze the different components of the immune system in a comprehensive fashion. Here, we demonstrate such an approach which provides information about total number of leukocytes, detailed quantitative and qualitative changes in the composition of lymphocyte subsets, cytokine levels in serum and functional properties of T cells, NK cells and monocytes. Using samples from a cohort of 24 healthy volunteers, we demonstrate the feasibility of our approach to detect changes in immune functions.

A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions.

The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

WhatSAP - 2B4 sends mixed messages in the absence of SAP.

2B4 (CD244), a member of the SLAM-related receptor family, has important immuno-regulatory functions including coactivating the cytotoxicity and cytokine secretion of NK cells. Immune modulation by 2B4 is dependent on the small intracellular signaling molecule SAP. In patients suffering from X-linked lymphoproliferative disease (XLP1), SAP is nonfunctional, not only abolishing the activating function of 2B4, but rendering this receptor inhibitory. In this issue of European Journal of Immunology, Meazza et al. [Eur. J. Immunol. 2014. 44: 1526-1534.] demonstrate that 2B4-mediated inhibition in NK cells from XLP1 patients is selective. While the activation of NK cells via ITAM-based receptors is blocked by inhibitory 2B4, DNAM-1, and NKG2D-dependent NK-cell activation is not affected by SAP deficiency. These findings provide an important insight into the different defective NK-cell functions in XLP1 patients and demonstrate the differential integration of redundant receptor signaling pathways in NK cells.

Surface CD107a/LAMP-1 protects natural killer cells from degranulation-associated damage.

Cytotoxic lymphocytes are important for immune responses against viral infections and cancer. They are able to kill target cells through the release of cytotoxic granules (CGs) without being harmed in the process. Because the lysosomal-associated membrane proteins (LAMPs) appear on the cell surface after CG exocytosis, we hypothesized that some of these proteins might be involved in transiently protecting cytotoxic lymphocytes from self-destruction. Intracellular expression of CD107a/LAMP-1, and to a lesser extent that of CD107b/LAMP-2, correlated with lymphocyte CG content. Engineered surface expression of CD107a/LAMP-1, but not of CD107b/LAMP-2, reduced the granule-mediated killing of transfected target cells. This was dependent on glycosylation of the CD107a/LAMP-1 hinge. Moreover, surface expression of CD107a/LAMP-1 reduced binding of perforin to cells. Importantly, knockdown of CD107a/LAMP-1 in primary human natural killer (NK) cells and deficiency of CD107a/LAMP-1 in mice resulted in increased NK cell apoptosis upon target cell-induced degranulation. Thus, our data support a novel role of CD107a/LAMP-1 in the protection of NK cells from degranulation-associated suicide, which may represent a general mechanism to transiently limit self-destruction by cytotoxic lymphocytes upon target cell killing.

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.

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.

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.

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.

Impact of Biological and Lifestyle Factors on Cognitive Aging and Work Ability in the Dortmund Vital Study: Protocol of an Interdisciplinary, Cross-sectional, and Longitudinal Study.

BACKGROUND: Previous research revealed several biological and environmental factors modulating cognitive functioning over a human's lifespan. However, the relationships and interactions between biological factors (eg, genetic polymorphisms, immunological parameters, metabolic products, or infectious diseases) and environmental factors (eg, lifestyle, physical activity, nutrition, and work type or stress at work) as well as their impact on cognitive functions across the lifespan are still poorly understood with respect to their complexity. OBJECTIVE: The goal of the Dortmund Vital Study is to validate previous hypotheses as well as generate and validate new hypotheses about the relationships among aging, working conditions, genetic makeup, stress, metabolic functions, the cardiovascular system, the immune system, and mental performance over the human lifespan with a focus on healthy working adults. The Dortmund Vital Study is a multidisciplinary study involving the Departments of Ergonomics, Immunology, Psychology and Neurosciences, and Toxicology at the Leibniz Research Centre for Working Environment and Human Factors at the Technical University of Dortmund (IfADo) in Germany, as well as several national and international partners. METHODS: The Dortmund Vital Study is designed as a combined cross-sectional and longitudinal study. Approximately 600 healthy subjects aged between 20 and 70 years will participate. A wide range of demographic, psychological, behavioral, sensory, cardiovascular, immunological, and biochemical data, a comprehensive electroencephalography (EEG)-based cognitive test battery as well as structural and functional magnetic resonance imaging (MRI) have been included in the study. RESULTS: The study was approved by the Ethics Committee of IfADo in October 2015. The baseline testing was conducted between 2016 and 2021 and will be repeated every 5 years (3 follow-up measures until 2035). As of March 2020 (until the outbreak of the COVID-19 pandemic), 593 participants have been enrolled. Some results from the cross-sectional part of the study were already published, further results will be published soon. Longitudinal data will be analyzed and published by 2025. CONCLUSIONS: We anticipate that the study will shed light on sources of interindividual differences in the alterations of cognitive functioning with increasing age and reveal biological and lifestyle markers contributing to work ability, longevity, and healthy aging on the one hand, and to risk factors for cognitive decline, mild cognitive impairment, or even dementia on the other hand. TRIAL REGISTRATION: ClinicalTrials.gov NCT05155397; https://clinicaltrials.gov/ct2/show/NCT05155397. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32352.

Results of the Optimune trial: A randomized controlled trial evaluating a novel Internet intervention for breast cancer survivors.

INTRODUCTION: After the acute treatment phase, breast cancer patients often experience low quality of life and impaired mental health, which could potentially be improved by offering cognitive behavioural therapy (CBT) and addressing exercise and dietary habits. However, CBT and other behavioural interventions are rarely available beyond the acute treatment phase. Internet-based interventions could bridge such treatment gaps, given their flexibility and scalability. In this randomized controlled trial (RCT), we investigated the effects of such an intervention ("Optimune") over three months. METHODS: This RCT included 363 female breast cancer survivors (age range = 30-70), recruited from the community, who had completed the active treatment phase. Inclusion criteria were: breast cancer diagnosis less than 5 years ago and acute treatment completion at least 1 month ago. Participants were randomly assigned to (1) an intervention group (n = 181), in which they received care as usual (CAU) plus 12-month access to Optimune immediately after randomization, or (2) a control group (n = 182), in which they received CAU and Optimune after a delay of 3 months. Primary endpoints were quality of life (QoL), physical activity, and dietary habits at three months. We hypothesized that intervention group participants would report better QoL, more physical activity, and improved dietary habits after 3 months. RESULTS: Intention-to-treat (ITT) analyses revealed significant effects on QoL (d = 0.27, 95% CI: 0.07-0.48) and dietary habits (d = 0.36, 95% CI: 0.15-0.56), but the effect on physical exercise was not significant (d = 0.30; 95% CI: 0.10-0.51). DISCUSSION: These findings suggest the effectiveness of Optimune, a new CBT-based Internet intervention for breast cancer survivors, in facilitating improvements in quality of life and dietary habits. Efforts to disseminate this intervention more broadly may be warranted. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03643640. Registered August 23rd 2018, https://clinicaltrials.gov/ct2/show/NCT03643640.

Low self-reported stress despite immune-physiological changes in paramedics during rescue operations.

Despite the high stress levels, paramedics seem to ignore or even negate the stress. This can be detrimental and lead to stress-related diseases. Therefore, we investigated the divergence between physiological and psychological stress responses of paramedics. Participants were 16 paramedics and 17 white-collar workers. We assessed psychological stress parameters, cortisol awakening response (CAR), and quantified immune parameters. In paramedics, electrocardiogram (ECG) was measured during one complete 24-hour shift. Our results revealed that CAR was higher in paramedics compared to controls. An alteration of immune parameters was observed even during days of free time. Also, ECG recordings showed acute stress in paramedics during rescue situations. Questionnaires revealed that rescue-service specific stressors affect psychological outcomes. However, paramedics reported significantly less mental stress and higher levels of depersonalization than controls. Taken together, our results suggest higher stress in paramedics compared to controls. However, paramedics negate their daily stress. Our findings underline therefore the importance to develop stress-management interventions for paramedics including sensitization for their stress reactions.