A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

Host immune responses and possible therapeutic targets for viral respiratory tract infections in susceptible populations: a narrative review.

BACKGROUND: Respiratory viruses are associated with significant global morbidity and mortality, as well as socioeconomic factors. Certain conditions and patient groups are more susceptible to develop severe viral respiratory tract infections (RTIs). OBJECTIVES: To summarise the data on deregulated immune pathways that have been associated with increased susceptibility to severe viral RTIs in certain populations. We also describe the commonalities of the defective immune pathways across these susceptible populations that may represent possible targets for future therapeutic or preventative approaches. SOURCES: We conducted free searches in Medline, Scopus, and Google Scholar for studies focusing on potential mechanisms of immune dysfunction that may be associated with severe viral RTIs in susceptible populations with conditions including pregnancy, obesity, diabetes mellitus, hypertension, cardiovascular disease, asthma, chronic obstructive pulmonary disease (COPD), chronic kidney disease, and extremes of age. We considered preclinical/animal data, original human studies, and reviews. CONTENT: Innate and adaptive immune responses become quantitatively and qualitatively compromised in aging, obesity, and diabetes mellitus, with the most pronounced changes affecting T cells. Moreover, immune dysregulation by the so-called inflamm-aging results in chronic low-grade inflammation in such conditions. Increased leptin levels affect the immune system particularly in obesity, while leptin dysregulation plays a role in asthma and COPD pathogenesis. Deficient production of interferon (IFN) type I and III in response to rhinovirus contributes to asthma exacerbations. Similar attenuation of IFN production in response to influenza and rhinovirus has been documented in pregnancy. Dampened type I IFN responses have also been found in diet-induced obese mice and in obese individuals. IMPLICATIONS: Immunosenescence and chronic low-grade inflammation accompanying aging and a variety of chronic conditions, such as diabetes, obesity, asthma, COPD, chronic renal disease, and hypertension, contribute to the poor outcomes observed following viral respiratory infections. Commonly affected pathways may represent potential future therapeutic targets.

Kinetics and Interrelations of the Renin Aldosterone Response to Acute Psychosocial Stress: A Neglected Stress System.

CONTEXT: The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular homeostasis and its dysfunction relates to negative health consequences. Acute psychosocial stress seems to activate the RAAS in humans, but stress kinetics and interrelations of RAAS parameters compared with a nonstress control group remain inconclusive. OBJECTIVE: We systematically investigated in a randomized placebo-controlled design stress kinetics and interrelations of the reactivity of RAAS parameters measured in plasma and saliva to standardized acute psychosocial stress induction. METHODS: 58 healthy young men were assigned to either a stress or a placebo control group. The stress group underwent the Trier Social Stress Test (TSST), while the control group underwent the placebo TSST. We repeatedly assessed plasma renin, and plasma and salivary aldosterone before and up to 3 hours after stress/placebo. We simultaneously assessed salivary cortisol to validate successful stress induction and to test for interrelations. RESULTS: Acute psychosocial stress induced significant increases in all endocrine measures compared with placebo-stress (all P ≤ .041). Highest renin levels were observed 1 minute after stress, and highest aldosterone and cortisol levels 10 and 20 minutes after stress, with salivary aldosterone starting earlier at 1 minute after stress. Renin completed recovery at 10 minutes, cortisol at 60 minutes, salivary aldosterone at 90 minutes, and plasma aldosterone at 180 minutes after stress. Stress increase scores of all endocrine measures related to each other, as did renin and cortisol areas under the curve with respect to increase (AUCi) and salivary and plasma aldosterone AUCi (all P ≤ .047). CONCLUSIONS: Our findings suggest that in humans acute psychosocial stress induces a differential and interrelated RAAS parameter activation pattern. Potential implications for stress-related cardiovascular risk remain to be elucidated.

Immunoproteasome Inhibition Impairs T and B Cell Activation by Restraining ERK Signaling and Proteostasis.

Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits.

Novel dengue virus NS2B/NS3 protease inhibitors.

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.