Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Specific T Cells and Antibodies in Coronavirus Disease 2019 (COVID-19) Protection: A Prospective Study.

BACKGROUND: During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. METHODS: In 5340 Moscow residents, we evaluated anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan-Meier estimator method, for up to 300 days postinclusion. RESULTS: We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. CONCLUSIONS: We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti-COVID-19 policies. Clinical Trials Registration. NCT04898140.

The Role of D2-like Dopaminergic Receptor in Dopamine-mediated Modulation of Th17-cells in Multiple Sclerosis.

BACKGROUND: Dopamine is one of the main mediators capable regulate the neuroimmune interaction and is involved in multiple sclerosis (MS) pathogenesis. OBJECTIVE: The aim of this study was to clarify the role of dopamine and its receptors in modulation of Th17-cells in MS. METHODS: 34 relapsing-remitting MS patients and 23 healthy subjects were examined. To assess the effect of dopamine on Th17-cells, CD4+ T-cells were cultured in the presence of dopamine and antagonist or agonist of D1- or D2-like dopaminergic receptors and stimulated with anti-CD3/CD28- microbeads. The levels of cytokines in supernatants were assessed by ELISA. RESULTS: Production of interleukin-17 (IL-17), interferon-γ (IFN-γ), granulocyte-colony stimulating factor (GM-CSF), and IL-21 by CD4+ T-cells as well as dopamine were comparable between the groups. Dopamine suppressed cytokine secretion by activated СD4+ T-cells in both groups. Blockade of D1-like dopaminergic receptor with a specific antagonist SCH23390 did not affect dopaminemediated cytokine suppression. In contrast, blockade of D2-like dopaminergic receptor by sulpiride decreased dopamine's inhibitory effect on IL-17 secretion in both groups and GM-CSF and IL-21 production in MS patients. Blockade of D1-like dopaminergic receptor directly inhibited IL-17, IFN- γ, GM-CSF in both groups and IL-21 production in healthy subjects, while blockade of D2-like dopaminergic receptor had no effect on cytokine secretion. Finally, activation of D2-like dopaminergic receptor with a specific agonist quinpirole decreased cytokine production in both groups. CONCLUSION: These data suggest an inhibitory role of dopamine on Th17-cells in MS, which could be mediated by the activation of the D2-like dopaminergic receptor.

Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKß or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

The influence of glatiramer acetate on Th17-immune response in multiple sclerosis.

Glatiramer acetate (GA) is approved for the treatment of multiple sclerosis (MS). However, the mechanism of action of GA in MS is still unclear. In particular, it is not known whether GA can modulate the pro-inflammatory Th17-type immune response in MS. We investigated the effects of original GA (Copaxone®, Teva, Israel) and generic GA (Timexone®, Biocad, Russia) on Th17- and Th1-type cytokine production in vitro in 25 patients with relapsing-remitting MS and 25 healthy subjects. Both original and generic GA at concentrations 50-200 µg/ml dose-dependently inhibited interleukin-17 and interferon-γ production by anti-CD3/anti-CD28-activated peripheral blood mononuclear cells from MS patients and healthy subjects. This effect of GA was reproduced using purified CD4+ T cells, suggesting that GA can directly modulate the functions of Th17 and Th1 cells. At high concentrations (100-200 µg/ml), GA also suppressed the production of Th17-differentiation cytokines (interleukin-1ß and interleukin-6) by lipopolysaccharide (LPS)-activated dendritic cells (DCs). These GA/LPS-treated DCs induced lower interleukin-17 and interferon-γ production by autologous CD4+ T cells compared to LPS-treated DCs. These data suggest that GA can inhibit Th17-immune response and that this inhibitory effect is preferentially exercised by direct influence of GA on T cells. We also demonstrate a comparable ability of original and generic GA to modulate pro-inflammatory cytokine production.

Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

The role of dopamine in modulation of Th-17 immune response in multiple sclerosis.

Neuromediators may modulate neuroinflammation, particularly in multiple sclerosis (MS). We investigated the effects of dopamine (DA) on the pro-inflammatory Th17-branch of immunity in 43 patients with relapsing-remitting MS and 20 healthy subjects. Serum DA was lower in MS relapse, whereas percentages of blood CD4(+)CD26(+)CD161(+)CD196(+) Th17-cells and production of interleukin-17 (IL-17) and interferon-gamma by anti-CD3/anti-CD28-stimulated peripheral blood mononuclear cells (PBMC) were higher in MS relapse than in remission or healthy subjects. DA suppressed IL-17 production by PBMC from MS patients and healthy subjects. The suppressive effect of DA was abolished in the presence of an antagonist of D2-like receptors (sulpiride). These data suggest an anti-inflammatory role for DA in MS.

Complexes of DNA with the Antimicrobial Peptide LL37 Augment NK Cell Functions by Inducing Type I Interferon Production from Circulating Monocytes and Plasmacytoid Predendritic Cells.

The cationic antimicrobial peptide, LL37, forms electrostatic complexes with DNA (LL37-DNA), which are potent activators of circulating plasmacytoid predendritic cells (ppDCs) and monocytes. However, the effects of LL37-DNA on other immune cell types, such as NK cells, are poorly characterized. In this study, we show that complexes of human genomic DNA (hgDNA) or synthetic double-stranded oligodeoxynucleotides with LL37 strongly enhance natural cytotoxicity of human peripheral blood mononuclear cells (PBMCs) upon an overnight culture, whereas hgDNA alone has no effect, and LL37 alone is moderately active. LL37-DNA complexes potentiate degranulation of, and interferon (IFN)-γ production by, NK cells upon subsequent encounter of K562 target cells. The complexes do not influence percentages of NK cells among PBMCs or the expression of cytotoxic proteins by NK cells. Using neutralizing anticytokine antibodies and immunomagnetic depletion of different subpopulations of PBMCs, we found that the effect of LL37-DNA on NK cells is indirect and mediated by type I IFNs produced by monocytes and, to a lesser extent, by ppDCs. We discuss possible roles of LL37-DNA complexes in the regulation of NK cell functions and in the treatment of cancer.

Reduced degranulation of NK cells in patients with frequently recurring herpes.

NK cells lyse virus-infected cells by degranulation; however, alterations in NK cell degranulation in persistent viral infections have not been directly studied. Earlier reports have documented a decrease in NK activity in patients with frequently recurring herpes (FRH). We corroborate these findings by showing that the degranulation responses of blood NK cells from patients with FRH, both during relapse and during remission, are significantly lower than those in healthy donors. The impaired degranulation was probably not caused by defective target cell recognition, since it was observed upon stimulation both with K562 cells and with a receptor-independent stimulus (phorbol 12-myristate 13-acetate plus ionomycin). We also show that the intracellular expression of perforin and CD107a by NK cells from patients with FRH is not different from that in healthy donors, thus excluding that the low NK cell degranulation in FRH is caused by a smaller size of the lytic granule compartment. We confirm previous reports on lowered NK activity in FRH patients and show that NK activity is significantly impaired only during remission, but not relapse; the causes for the discrepancy between the low degranulation and "normal" NK cell activity during relapse are discussed. In all, these data point at the deficit of NK cell degranulation in FRH. Whether this is a predisposing factor or a consequence of herpes simplex virus infection requires further investigation.

Muropeptides trigger distinct activation profiles in macrophages and dendritic cells.

Bacterial peptidoglycan and its muropeptide derivatives potently activate mammalian innate immune system and are promising immunomodulators and vaccine adjuvants. However, their effects on human antigen-presenting cells, such as dendritic cells (DCs) and Mphi, are not fully understood. Lysozyme treatment of PG from Salmonella typhi yielded three muropeptides, GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP (GM-3P), GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP-D-Ala (GM-4P), and a dimer (GM-4P)(2), in which two GM-4P monomers are linked through their peptidic moieties. All three muropeptides induced TNF-alpha and IL-6 production by Mphi (GM-3P>GM-4P>>(GM-4P)(2)), but failed to trigger TNF-alpha, IL-6 and IL-12p70 production by immature DCs. At the same time, muropeptide-stimulated DCs abundantly produced inflammatory chemokines IL-8, MIP-1 alpha and MIP-1 beta, as well as displayed signs of phenotypic and functional maturation. Thus, muropeptide-dependent pro-inflammatory cytokine production is repressed in DCs. While this defect may be partly compensated in vivo by muropeptide-activated Mphi, neither Mphi nor DCs produce Th1- or Th17-polarizing cytokines upon muropeptide stimulation, which may contribute to the preferential induction of Th2 responses by muropeptides and should be taken into account when designing muropeptide-based immunomodulators and adjuvants.