M ycobacterium tuberculosis-specific CD4 T-cell scoring discriminates tuberculosis infection from disease.

BACKGROUND: Rapid and reliable diagnostic work-up of tuberculosis (TB) remains a major healthcare goal. In particular, discrimination of TB infection from TB disease with currently available diagnostic tools is challenging and time consuming. This study aimed at establishing a standardised blood-based assay that rapidly and reliably discriminates TB infection from TB disease based on multiparameter analysis of TB antigen-reactive CD4+ T-cells acting as sensors for TB stage-specific immune status. METHODS: 157 HIV-negative subjects with suspected TB infection or TB disease were recruited from local tertiary care hospitals in Berlin (Germany). Peripheral blood mononuclear cells were analysed for CD4+ T-cells reactive to the Mycobacterium tuberculosis antigens purified protein derivative and early secretory antigenic target 6 kDa/culture filtrate protein 10. The activation state of TB antigen-reactive T-cells, identified by surface expression of CD154, was evaluated according to the expression profile of proliferation marker Ki-67 and activation markers CD38 and HLA-DR. Using data from 81 subjects with clinically confirmed TB infection (n=34) or culture-proven pulmonary or extrapulmonary TB disease (n=47), 12 parameters were derived from the expression profile and integrated into a scoring system. RESULTS: Using the scoring system, our assay (TB-Flow Assay) allowed reliable discrimination of TB infection from both pulmonary and extrapulmonary TB disease with high sensitivity (90.9%) and specificity (93.3%) as was confirmed by Monte-Carlo cross-validation. CONCLUSION: With low time requirement, ease of sample collection, and high sensitivity and specificity both for pulmonary and extrapulmonary TB disease, we believe this novel standardised TB-Flow Assay will improve the work-up of patients with suspected TB disease, supporting rapid TB diagnosis and facilitating treatment decisions.

Gut microbiota dysbiosis is associated with altered tryptophan metabolism and dysregulated inflammatory response in COVID-19.

The clinical course of COVID-19 is variable and often unpredictable. To test the hypothesis that disease progression and inflammatory responses associate with alterations in the microbiome and metabolome, we analyzed metagenome, metabolome, cytokine, and transcriptome profiles of repeated samples from hospitalized COVID-19 patients and uninfected controls, and leveraged clinical information and post-hoc confounder analysis. Severe COVID-19 was associated with a depletion of beneficial intestinal microbes, whereas oropharyngeal microbiota disturbance was mainly linked to antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine and reduced levels of several other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Moreover, reduced concentrations of various tryptophan metabolites were associated with depletion of Faecalibacterium, and tryptophan decrease and kynurenine increase were linked to enhanced production of inflammatory cytokines. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.

A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4+ T cells and is defective in Crohn´s disease patients.

Immunosuppressive Interleukin (IL)-10 production by pro-inflammatory CD4+ T cells is a central self-regulatory function to limit aberrant inflammation. Still, the molecular mediators controlling IL-10 expression in human CD4+ T cells are largely undefined. Here, we identify a Notch/STAT3 signaling-module as a universal molecular switch to induce IL-10 expression across human naïve and major effector CD4+ T cell subsets. IL-10 induction was transient, jointly controlled by the transcription factors Blimp-1/c-Maf and accompanied by upregulation of several co-inhibitory receptors, including LAG-3, CD49b, PD-1, TIM-3 and TIGIT. Consistent with a protective role of IL-10 in inflammatory bowel diseases (IBD), effector CD4+ T cells from Crohn's disease patients were defective in Notch/STAT3-induced IL-10 production and skewed towards an inflammatory Th1/17 cell phenotype. Collectively, our data identify a Notch/STAT3-Blimp-1/c-Maf axis as a common anti-inflammatory pathway in human CD4+ T cells, which is defective in IBD and thus may represent an attractive therapeutic target.

Human immature myeloid dendritic cells trigger a TH2-polarizing program via Jagged-1/Notch interaction.

BACKGROUND: The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear. OBJECTIVE: Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). METHODS: IL-4 and IFN-gamma production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. RESULTS: Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype. CONCLUSION: CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components.

siRNA stabilization prolongs gene knockdown in primary T lymphocytes.

RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.