Intracellular Cytokine Production Research Articles

T cell immune discriminants of HIV reservoir size in a pediatric cohort of perinatally infected individuals.

The size of the latent HIV reservoir is associated with the timing of therapeutic interventions and overall health of the immune system. Here, we demonstrate that T cell phenotypic signatures associate with viral reservoir size in a cohort of HIV vertically infected children and young adults under durable viral control, and who initiated anti-retroviral therapy (ART) <2 years old. Flow cytometry was used to measure expression of immune activation (IA), immune checkpoint (ICP) markers, and intracellular cytokine production after stimulation with GAG peptides in CD4 and CD8 T cells from cross-sectional peripheral blood samples. We also evaluated the expression of 96 genes in sort-purified total CD4 and CD8 T cells along with HIV-specific CD4 and CD8 T cells using a multiplexed RT-PCR approach. As a measure of HIV reservoir, total HIV-DNA quantification by real-time PCR was performed. Poisson regression modeling for predicting reservoir size using phenotypic markers revealed a signature that featured frequencies of PD-1+CD4 T cells, TIGIT+CD4 T cells and HIV-specific (CD40L+) CD4 T cells as important predictors and it also shows that time of ART initiation strongly affects their association with HIV-DNA. Further, gene expression analysis showed that the frequencies of PD-1+CD4 T cells associated with a CD4 T cell molecular profile skewed toward an exhausted Th1 profile. Our data provide a link between immune checkpoint molecules and HIV persistence in a pediatric cohort as has been demonstrated in adults. Frequencies of PD-1+ and TIGIT+CD4 T cells along with the frequency of HIV-specific CD4 T cells could be associated with the mechanism of viral persistence and may provide insight into potential targets for therapeutic intervention.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Co-expression of NKp46 with activating or inhibitory receptors on, and cytokine production by, uterine endometrial NK cells in recurrent pregnancy loss

NKp46 (CD335) is one of the activating receptors expressed on NK cells and its expression is decreased in patients with reproductive failure. However, the reasons remain unknown. In this study, we aimed to clarify the significance of decreased NKp46 expression in reproductive failure. Uterine endometrial samples collected from 39 patients with recurrent pregnancy loss (RPL) were assigned to high- or low-risk groups based on an 18 % ratio of CD16+/CD56dim NK cells in uterine endometrial NK (uNK) cells. We analyzed the expression of NKp46 and other activating or inhibitory receptors on, and intracellular cytokine production by NK cells using multicolor flow cytometry. The numbers of NKp46+/CD16− NK, NKp46+/NKG2C− NK, IL-4+/CD56+ NK, and IL-10+/CD56+ NK cells were significantly decreased, whereas that TNF-α+/CD56+ NK cells was significantly increased in the high-risk group, when compared with the low-risk group (P < 0.05 for all). The ratios of TNF-α/IL-4, IFN-γ/IL4, TNF-α/IL-10, and IFN-γ/IL10 cytokine production in uNK cells were significantly increased in the high-risk when compared with the low-risk group (P < 0.05, for all). It is suggested that low expression of activating receptors on NKp46 uNK cells is more prevalent in high-risk women.

Successful Direct Acting Antiviral Therapy in Chronic Hepatitis C Normalizes IFNγ and IL2 Production in T Cells Together with TLR8 Expression and Functionality in Peripheral Blood Mononuclear Cells.

Chronic hepatitis C infection (HCV) activates a systemic cell-mediated immune response characterized by the production of IFNγ and an innate immune response addressed by the activation of TLR signaling. We aimed to investigate whether HCV eradication by direct acting antivirals (DAA) leads to a recovery in cell-mediated immune response and TLR expression and functionality. Blood samples were obtained in HCV infected patients before DAA treatment and at week +48 after the end of treatment. Results were compared to healthy controls. Cell surface expression of TLR8 was assessed on peripheral blood mononuclear cells (PBMCs) by flow cytometry. Freshly isolated PBMCs were cultured with specific TLR8 agonists and intracellular production of cytokines was determined by flow-cytometry after ex vivo TLR8 activation with ssRNA 40. Production of IFNγ, IL2 and IL17 was assessed by flow cytometry in T cells after polyclonal activation. Included were 50 HCV-infected patients and 15 controls. TLR8 expression in PBMCs was significantly increased before treatment and recovered normal levels at week +48. Production of IL1b, IL6 and TNFα dependent on the activation of TLR8 in PBMCs was also increased in patients before DAA treatment, with a significant reduction at week +48. Combined expression of IFNγ and IL2 in CD4+ T cells in HCV-infected patients was significantly increased compared to controls and recovered normal levels at week +48. DAA-mediated clearance of HCV is associated with a decreased expression and activation of TLR8 in PBMCs until healthy control levels which is accompanied by a reduction in the Th1 response.

Exercise-induced modulation of monocytes in breast cancer survivors

BackgroundExercise training reduces inflammation in breast cancer survivors; however, the mechanism is not fully understood. ObjectivesThe effects of acute and chronic exercise on monocyte toll-like receptor (TLR2 and 4) expression and intracellular cytokine production were examined in sedentary breast cancer survivors. MethodsEleven women with stage I, II, or III breast cancer within one year of treatment completion performed an acute, intermittent aerobic exercise trial. Blood samples were obtained before, immediately, and 1 h after a 45-min acute exercise trial that was performed before and after 16 weeks of combined aerobic and resistance. LPS-stimulated intracellular IL-1ß, TNF, and IL-6 production, and TLR2 and TLR4 expression were evaluated in CD14+CD16- and CD14+CD16+ monocytes using flow cytometry. ResultsExercise training decreased IL-1ß+CD14+CD16- proportion (24.6%, p=0.016), IL-1ß+CD14+CD16- mean fluorescence intensity (MFI) (−9989, p=0.014), IL-1ß+CD14+CD16+ MFI (−11101, p=0.02), and IL-6+CD14+CD16- proportion (16.9%, P=0.04). TLR2 and TLR4 expression did not change following exercise training but decreased 1 h after acute exercise in CD14+CD16- (−63, p=0.002) and CD14+CD16+ (−18, p=0.006) monocytes, respectively. Immediately after the acute exercise, both monocyte subgroup cell concentration increased, with CD14+CD16+ concentrations being decreased at 1 h post without changes in intracellular cytokine production. ConclusionsExercise training reduced monocyte intracellular pro-inflammatory cytokine production, especially IL-1ß, although these markers did not change acutely. While acute exercise downregulated the expression of TLR2 and TLR4 on monocytes, this was not sustained over the course of training. These results suggest that the anti-inflammatory effect of combined aerobic and resistance exercise training in breast cancer survivors may be, in part, due to reducing resting monocyte pro-inflammatory cytokine production.

Low-Dose Decitabine Augments the Activation and Anti-Tumor Immune Response of IFN-γ+ CD4+ T Cells Through Enhancing IκBα Degradation and NF-κB Activation

BackgroundCD4+ T cells play multiple roles in controlling tumor growth and increasing IFN-γ+ T-helper 1 cell population could promote cell-mediated anti-tumor immune response. We have previously showed that low-dose DNA demethylating agent decitabine therapy promotes CD3+ T-cell proliferation and cytotoxicity; however, direct regulation of purified CD4+ T cells and the underlying mechanisms remain unclear.MethodsThe effects of low-dose decitabine on sorted CD4+ T cells were detected both in vitro and in vivo. The activation, proliferation, intracellular cytokine production and cytolysis activity of CD4+ T cells were analyzed by FACS and DELFIA time-resolved fluorescence assays. In vivo ubiquitination assay was performed to assess protein degradation. Moreover, phosphor-p65 and IκBα levels were detected in sorted CD4+ T cells from solid tumor patients with decitabine-based therapy.ResultsLow-dose decitabine treatment promoted the proliferation and activation of sorted CD4+ T cells, with increased frequency of IFN-γ+ Th1 subset and enhanced cytolytic activity in vitro and in vivo. NF-κB inhibitor, BAY 11-7082, suppressed decitabine-induced CD4+ T cell proliferation and IFN-γ production. In terms of mechanism, low-dose decitabine augmented the expression of E3 ligase β-TrCP, promoted the ubiquitination and degradation of IκBα and resulted in NF-κB activation. Notably, we observed that in vitro low-dose decitabine treatment induced NF-κB activation in CD4+ T cells from patients with a response to decitabine-primed chemotherapy rather than those without a response.ConclusionThese data suggest that low-dose decitabine potentiates CD4+ T cell anti-tumor immunity through enhancing IκBα degradation and therefore NF-κB activation and IFN-γ production.

An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.

Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

Functional Activity of Non-Proliferating Mesenchymal Stromal Cells Cultured at Different Densities.

We analyzed the state of intracellular compartments and production of cytokines in MSC depending on the culture density. MSC were growth-arrested with mitomycin C and seeded at a density of 300-7000 cell/cm2. MSC in low-density cultures had 2-fold higher levels of transmembrane mitochondrial potential (MitoTracker Red) and endogenous ROS (CMH2DCFDA), lysosomal compartments were less acidified (LysoTracker Green DND26), the production of immunoregulatory and angiogenic mediators VEGF, IL-6, IL-8, MCP-1, TGF-β was more intensive. It was assumed that culture density can be an effective tool for phenotypic polarization of MSC providing directional changes in their properties.

Skewed intracellular cytokine production of iNKT cells toward Th2-related responses in alloimmunized thalassemia patients

IntroductionRed blood cell alloimmunization is a challenging issue in thalassemia patients. Several studies have investigated the role of different immune system compartment in alloimmunization, but the exact mechanism remains unclear. Considering the immunoregulatory function of iNKT cells and their subsets, in this study, we evaluated the possible role of these cells in alloimmunization status of thalassemia patients. Methods78 β-thalassemia major patients (41 alloimmunized and 37 non-alloimmunized) and 17 healthy controls were engaged in this study. Mononuclear cells were isolated from peripheral blood samples and stimulated for cytokine production. Samples were subjected to flow cytometry for enumeration of iNKT cells and characterized based on their cytokine production pattern. Finally, the results correlated with alloimmunization status, clinical and laboratory data. ResultsResults demonstrated that the number of iNKT, iNKT+IFN-ɤ+, and iNKT+IL-4+ cells in thalassemia group was significantly higher than healthy controls while no significant change was observed in the number of these cells between alloimmunized and non-alloimmunized thalassemia patients. Interestingly, the ratio of iNKT+IL-4+: iNKT+IFN-γ+ cells in alloimmunized thalassemia group represent a considerable increase in comparison to both non-alloimmunized thalassemia group and healthy controls. However, evaluating this value in non-alloimmunized group represents an approximately equal ratio of 0.94, which was almost similar to this ratio in the control group (0.99).Conclusion.Our results illustrated a noteworthy imbalance in the ratio of iNKT cell subsets in favour of IL-4 producing iNKT cells in alloimmunized thalassemia patients. Regarding the role of IL-4 in stimulating the Th2-related immune responses, this imbalance could consider as a possible mechanism in alloantibody responses of thalassemia patients.

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells.

BackgroundMethylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells.MethodsMC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured.ResultsPretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone.ConclusionSpironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Polysaccharide from Astragalus membranaceus promotes the activation of human peripheral blood and mouse spleen dendritic cells.

Astragalus membranaceus (A. membranaceus) is a widely used traditional herb in China and Korea. A. membranaceus polysaccharides (AMP), which make up a major part of the root extract, have been shown to modulate immune modulations, especially activation of bone marrow-derived dendritic cells (BMDCs) and T cells. However, the immune stimulatory effect of AMP in the mouse in vivo and human peripheral blood DCs (PBDCs) has not been well investigated. In this study, we found that intravenous (i.v.) injection of AMP in C57BL/6 mice induced remarkable elevations in co-stimulatory and MHC class I and II molecule levels in the splenic DCs and its subsets. The stimulatory effect of DCs by AMP was elevated 6 h after treatment, which rapidly decreased 18 h after injection. Furthermore, AMP promoted intracellular production of pro-inflammatory cytokines in spleen DC subsets, which contributed elevation of serum cytokine levels. Finally, the AMP promoted PBDC activation. Thus, these results demonstrate that AMP can be used as an immune stimulatory molecules in human and mouse.

The effect of glucocorticoids in combination with azithromycin or theophylline on cytokine production by NK and NKT-like blood cells of patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by reduced sensitivity of cells to the anti-inflammatory effects of glucocorticoids (GCs). Azithromycin and a low dose theophylline have a significant impact on molecular mechanisms leading to corticosteroid resistance. The aim of this study was to evaluate the ability of azithromycin and theophylline to enhance the anti-inflammatory effects of GCs on the production of cytokines by NK and NKT-like blood cells of COPD patients. Whole blood cells from COPD patients (n=21) were incubated in the presence of budesonide (10 nM), azithromycin (10 μg/mL), theophylline (1 μM), or their combinations and stimulated with phorbol myristate acetate (50 ng/mL). Intracellular production of proinflammatory cytokines in NK (CD3-CD56+) and NKT-like (CD3+CD56+) blood cells was analyzed by flow cytometry. Budesonide reduced synthesis of interleukin 4 (IL-4), CXCL8, tumor necrosis factor α (TNFα) by NK and NKT-like cells, as well as production of interferon γ (IFNγ) by NK cells. Azithromycin suppressed production of IL-4 and CXCL8 by NK and NKT-like cells, and theophylline inhibited IL-4 synthesis by these lymphocytes. The combination of azithromycin and budesonide had a more pronounced inhibitory effect on the production of IL-4 and CXCL8 by NK and NKT-like cells than the effect of these drugs alone. The combination of theophylline and budesonide suppressed synthesis of IL-4 and CXCL8 by NK and NKT-like cells, as well as the production of TNFα and IFNγ by NK cells stronger than budesonide alone. The obtained results demonstrate the benefits for the combined use of GCs with theophylline at a low dose or azithromycin to suppress the inflammatory process in patients with COPD.

Radioprotective Effect of Flavonoids on Ionizing Radiation-Induced Brain Damage.

Patients receiving brain radiotherapy may suffer acute or chronic side effects. Ionizing radiation induces the production of intracellular reactive oxygen species and pro-inflammatory cytokines in the central nervous system, leading to brain damage. Complementary Chinese herbal medicine therapy may reduce radiotherapy-induced side effects. Flavonoids are a class of natural products which can be extracted from Chinese herbal medicine and have been shown to have neuroprotective and radioprotective properties. Flavonoids are effective antioxidants and can also inhibit regulatory enzymes or transcription factors important for controlling inflammatory mediators, affect oxidative stress through interaction with DNA and enhance genomic stability. In this paper, radiation-induced brain damage and the relevant molecular mechanism were summarized. The radio-neuro-protective effect of flavonoids, i.e., antioxidant, anti-inflammatory and maintaining genomic stability, were then reviewed. We concluded that flavonoids treatment may be a promising complementary therapy to prevent radiotherapy-induced brain pathophysiological changes and cognitive impairment.

Comprehensive Phenotyping of Peripheral Blood T Lymphocytes in Healthy Mice.

T lymphocytes play a central role in antigen-specific immune responses. They modulate the function of different immune cells both through a direct contact (receptor binding) and through the secretion of cytokines. At the same time, they are deeply involved in the direct killing of aberrant target cells. T lymphocytes derive from a bone marrow precursor that migrates in the thymus where the main differentiation steps take place. Mature CD4 and CD8 single-positive cells, then, leave the thymus to reach the secondary lymphoid organs. T-cell subsets and their maturation steps can be identified mainly based on the expression of extracellular markers, intracellular transcription factors and cytokine production profiles. In this review, we report, from a cytometric point of view, an overview of the most important T-cell subpopulations and their differentiation state. © 2020 International Society for Advancement of Cytometry.

Streptococcus mutans bystander-induced bioeffects following sonodynamic antimicrobial chemotherapy through sonocatalytic performance of Curcumin-Poly (Lactic-co-Glycolic Acid) on off-target cells.

To assessed the Streptococcus mutans bystander-induced bioeffects following sonodynamic antimicrobial chemotherapy (SACT) by Curcumin-Poly (Lactic-co-Glycolic Acid) nanoparticles (Cur-PLGA-NPs). Cur-PLGA-NPs were synthesized and characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), and Attenuated Total Reflection Fourier Transform IR (ATR-FTIR) spectroscopy, as well as, determination of in vitro drug release. Following the successful synthesis and characterization of Cur-PLGA-NPs, the cell survival, intracellular ROS production, apoptotic effects, DNA fragmentation, and gene expression levels of pro-inflammatory cytokines were investigated on human gingival fibroblast (HGF) cells as off-target cells through S. mutans bystander-induced bioeffects following SACT (BCSS). No significant cytotoxic and damage caused by the release of ROS from BCSS were observed in HGF cells (P > 0.05). There was no DNA fragmentation and anti-proliferation effects on HGF cells. The expression levels of bFGF, TNF-α, and IL-8 genes were increased after exposure to BCSS to 15.4-, 13.5-, and 8.7-fold, respectively (P < 0.05), while TGF-ß and IL-10 were downregulated to -4.1- and -6.8-fold, respectively (P < 0.05). It could be concluded that there were no bystander bioeffects of targeted sonocatalytic stress on off-target cells.

Chilblain-like lesions and COVID-19 infection: A prospective observational study at Spain's ground zero

Chilblain-like lesions and COVID-19 infection: A prospective observational study at Spain's ground zero

TLR4 inhibition ameliorates mesencephalic substantia nigra injury in neonatal rats exposed to lipopolysaccharide via regulation of neuro-immunity

Dopaminergic neurons play a key role in motor function of the extrapyramidal system. Previous studies have shown that septic neonates may have cognitive dysfunction in later life. Toll-like receptor 4 (TLR4) is a transmembrane protein which is widely distributed in nerve cells. Its activation will activate the innate immune system through increasing an intracellular signaling pathway NF-κB and inflammatory cytokine production. Here, One-day-old rats were exposed to lipopolysaccharide (LPS, 1 mg/kg) as a sepsis model. We pre-treat neonatal rats with TAK-242, a TLR4 inhibitor, 1 day and 1 h before LPS injection to observe the neuroprotective effects on dopaminergic neurons of inhibition TLR4 in septic neonatal rats. The number of dopaminergic neurons in the mesencephalic substantia nigra in septic brain was significantly reduced. Meanwhile, the number of activated microglia was increased. Additionally, the total number of lattice in the open field test of septic rats was less than that of control rats at the age of postnatal day 80 (PND80), the total time of septic rats to catch the rope in the grip traction test was less than the control rats at the age of PND80, and the septic rats were easier to slide down in the slopes experiment. However, TAK-242 administration significantly ameliorated LPS-induced dopaminergic neurons loss, prevented microglia activation, and improved behavioral test results of septic rats. We also found that TLR4 inhibition decreased the LPS-induced high expressions of NF-κB p65 and IL-1β of microglia. In conclusion, TLR4 inhibition showed neuroprotective effects on septic neonatal rats via regulation of neuro-immunity.

Cow milk exosomes activate NK cells and γδT cells in human PBMCs in vitro

Cow milk is a nourishing food containing numerous essential nutrients. In Japan, the consumption of cow milk is thought to enhance resistance to exhaustion-related diseases. Although several nutrients in cow milk, such as lactoferrin, are thought to modulate immune cells, the mechanisms remain unclear. Recently, the immunoregulatory functions of food-derived microRNAs or exosomes have been reported. Therefore, we studied the effects of exosomes derived from cow milk (CM-Exs) on immune cells in the present study. We obtained blood samples from healthy adult donors with the approval of the ethics committee. Peripheral blood mononuclear cells (PBMCs) were stimulated with CM-Exs in the absence or presence of interleukin-2 (IL-2) and IL-12. Cell surface markers and intracellular cytokine production were analysed by flow cytometry. CM-Ex stimulation enhanced the expression of CD69 on NK cells. Although CM-Ex stimulation alone did not induce interferon-γ (IFN-γ) production by NK cells or γδT cells, simultaneous stimulation with CM-Ex, IL-2 and IL-12 significantly enhanced IFN-γ production. In conclusion, cow milk consumption alone may not activate immune cells; however, CM-Exs could enhance immune cells under inflammatory conditions.

Anti-CD20 rituximab IgG1, IgG3, and IgG4 but not IgG2 subclass trigger Ca2+ mobilization and cytotoxicity in human NK cells.

NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc-gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell-mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low-affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high-affinity (VV) and VF SNP variants was two- to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low-affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell-mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy.

IgG from atopic individuals can mediate non-atopic infant thymic and adult peripheral CD8+ TC2 skewing without influence on TC17 or TC22 cells.

The potential of IgG antibodies as allergy regulators has been discussed for decades and was brought to light that anti-allergen IgG is related to allergy inhibition in children during the first years of life and that IgG repertoire can differ between atopic and non-atopic individuals. Here, we aimed to evaluate in vitro the differential effects of purified IgG from atopic and non-atopic individuals on the production of IL-4, IL-17, and IL-22 by human intra-thymic and mature peripheral CD8+ T cells respectively termed as TC2, TC17, and TC22 cells. We additionally evaluated the IFN-􀁊 production by CD8+ T cells. Thereupon we used infants thymic tissues from non-atopic mothers and blood samples from individuals clinically classified as non-atopic. Thymocytes or PBMCs were cultured with IgG from atopic or non-atopic individuals. As controls, we used commercial IgG (Intravenous immunoglobulin - IVIg) or mock condition. The phenotype and intracellular cytokine production were evaluated using flow cytometry. IgG from atopic individuals could increase the frequency of TC2 cells in non-atopic infant thymic and adult peripheral cell cultures compared to all control conditions. Due to the TC2 cell's potential to collaborate with pathology and severity of asthma in humans, this evidence can cooperate with the understanding of the development of an atopic state.