Inhibition of glucose uptake in NK cells enhances their serial killing capacity

Glucose-transporter (GLUT)-inhibitors, like Glutor and Glupin, effectively inhibit the proliferation of different tumor cells, making them potential candidates for cancer therapies. Therefore, assessing the impact of GLUT-inhibitors on NK cell function is crucial, as these cells play an important role in anti-tumor response. Seahorse analysis of the energetic phenotype of NK cells treated with Glutor or Glupin showed decreased glycolysis. To further examine the effect of both inhibitors on NK cell effector functions, resting or pre-activated human NK cells were stimulated through CD16 in the presence or absence of Glutor or Glupin. This acute inhibition of the GLUT had no significant effect on NK cell cytotoxicity, cytokine secretion or killing capacity against tumor cells. To analyze possible long-term effects, we cultured freshly isolated NK cells for 3 weeks in the presence or absence of Glutor or Glupin. We could detect a lack of proliferation in case of Glutor-treatment, whereas Glupin-treated NK cells displayed a delayed proliferation. Analysis of various surface receptors showed that long-term treatment with Glutor or Glupin led to an altered NK cell phenotype compared to the control. Furthermore, we examined the cytotoxic and immunoregulatory function of these NK cells: Long-term treatment with Glutor reduced the degranulation and IFN- secretion after stimulation via CD16 or NKp30. Interestingly, Glupin did not affect degranulation in comparison to the control cells, whereas the IFN- secretion was significantly diminished after stimulation via CD16 or NKp30. Furthermore, the serial-killing capacity of long-term treated NK cells with Glupin was higher than that of control cells. Experiments regarding the usage of other fuels, like glutamine or fatty acids, revealed that NK cells did not use fatty acids to fulfil their functions and that glutamine seems to be not responsible for this increased serial killing capacity. RNA-sequencing data together with the analysis of NAD+/NADH concentrations of Glupin-treated NK cells suggests a possible role of CD38 and NAD+ in the serial killing capacity of NK cells. Further, RNA-sequencing of Glutor-treated NK cells displayed a cell cycle arrest, which could explain the lack of proliferation. These data identify Glupin as a suitable candidate for cancer therapy

Immunological fingerprint in coronavirus disease-19 convalescents with and without post-COVID syndrome

BackgroundSymptoms lasting longer than 12  weeks after severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection are called post-coronavirus disease (COVID) syndrome (PCS). The identification of new biomarkers that predict the occurrence or course of PCS in terms of a post-viral syndrome is vital. T-cell dysfunction, cytokine imbalance, and impaired autoimmunity have been reported in PCS. Nevertheless, there is still a lack of conclusive information on the underlying mechanisms due to, among other things, a lack of controlled study designs.MethodsHere, we conducted a prospective, controlled study to characterize the humoral and cellular immune response in unvaccinated patients with and without PCS following SARS-CoV-2 infection over 7 months and unexposed donors.ResultsPatients with PCS showed as early as 6 weeks and 7 months after symptom onset significantly increased frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells secreting IFNγ, TNF, and expressing CD40L, as well as plasmacytoid dendritic cells (pDC) with an activated phenotype. Remarkably, the immunosuppressive counterparts type 1 regulatory T-cells (TR1: CD49b/LAG-3+) and IL-4 were more abundant in PCS+.ConclusionThis work describes immunological alterations between inflammation and immunosuppression in COVID-19 convalescents with and without PCS, which may provide potential directions for future epidemiological investigations and targeted treatments

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

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

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 todetect antibodies directed against the receptor binding domain (RBD) of SARS-CoV-2 Spike, we identified individuals withSARS-CoV-2 infection after an outbreak at a local health institution. All but one COVID-19 patient developed detectableanti-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 theseindividuals 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 twovaccine doses for recovered individuals

DataSheet_1_15-month post-COVID syndrome in outpatients: Attributes, risk factors, outcomes, and vaccination status - longitudinal, observational, case-control study.pdf

BackgroundWhile the short-term symptoms of post-COVID syndromes (PCS) are well-known, the long-term clinical characteristics, risk factors and outcomes of PCS remain unclear. Moreover, there is ongoing discussion about the effectiveness of post-infection vaccination against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to aid in PCS recovery.MethodsIn this longitudinal and observational case-control study we aimed at identifying long-term PCS courses and evaluating the effects of post-infection vaccinations on PCS recovery. Individuals with initial mild COVID-19 were followed for a period of 15 months after primary infection. We assessed PCS outcomes, distinct symptom clusters (SC), and SARS-CoV-2 immunoglobulin G (IgG) levels in patients who received SARS-CoV-2 vaccination, as well as those who did not. To identify potential associating factors with PCS, we used binomial regression models and reported the results as odds ratios (OR) with 95% confidence intervals (95%CI).ResultsOut of 958 patients, follow-up data at 15 month after infection was obtained for 222 (23.2%) outpatients. Of those individuals, 36.5% (81/222) and 31.1% (69/222) were identified to have PCS at month 10 and 15, respectively. Fatigue and dyspnea (SC2) rather than anosmia and ageusia (SC1) constituted PCS at month 15. SARS-CoV-2 IgG levels were equally distributed over time among age groups, sex, and absence/presence of PCS. Of the 222 patients, 77.0% (171/222) were vaccinated between 10- and 15-months post-infection, but vaccination did not affect PCS recovery at month 15. 26.3% of unvaccinated and 25.8% of vaccinated outpatients improved from PCS (p= .9646). Baseline headache (SC4) and diarrhoea (SC5) were risk factors for PCS at months 10 and 15 (SC4: OR 1.85 (95%CI 1.04-3.26), p=.0390; SC5: OR 3.27(95%CI 1.54-6.64), p=.0009).ConclusionBased on the specific symptoms of PCS our findings show a shift in the pattern of recovery. We found no effect of SARS-CoV-2 vaccination on PCS recovery and recommend further studies to identify predicting biomarkers and targeted PCS therapeutics.</p