Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Abstract Background The murine discs large homolog 2 (DLG2; post synaptic density 93 (PSD-93); Chapsyn-110) is a member of the membrane-associated guanylate kinase (MAGUK) protein family involved in receptor assembly and associated with signaling enzymes on cell membranes. In neurons, DLG2 protein isoforms derived from alternatively spliced transcripts have been described to bind to NMDA (N-methyl-aspartate) receptors and K channels and to mediate clustering of these channels in the postsynaptic membrane. In myeloid cells of the immune system, such as dendritic cells (DCs), a lack of data exists on the expression or function of DLG2. In cDNA microarray transcriptome analyses, we found Dlg2 highly expressed in a subpopulation of plasmacytoid DCs (pDCs) stimulated to produce type I interferons (IFNs) such as IFNβ. Results Using RACE- and RT-PCR as well as immunoprecipitation followed by Western blotting we characterised the differential expression of the Dlg2 splice variants in IFNβ-producing pDCs. Besides Dlg2ɣ this cell population expressed a novel short Dlg2η transcript we termed Dlg2η3. Our expression data were integrated into information from genome databases to obtain a novel and comprehensive overview of the mouse Dlg2 gene architecture. To elucidate the intracellular localisation pattern of protein isoforms, ectopical expression analysis of fluorescently tagged DLG2 splice variants was performed. Here we found an enrichment of the larger isoform DLG2α1 at the plasma membrane while the newly identified shorter (DLG2η) isoform as well as DLG2ɣ were equally distributed throughout the cytoplasm. Additionally, DLG2η was also found in the nucleus. Analysis of Dlg2-knockout mice previously generated by deleting exon 9 surprisingly revealed that the protein for the novel DLG2η isoform was still expressed in the brain and in bone marrow-derived pDCs from mice carrying the homozygous deletion (Dlg2 ΔE9/ΔE9 ). Conclusion We describe a novel splice variant of the mouse Dlg2 gene termed Dlg2η and define the differential expression pattern of DLG2 isoforms in IFNβ-producing pDCs. The presence of DLG2η protein in the CNS of Dlg2 ΔE9/ΔE9 mice might influence the phenotype of these mice and has to be taken into account in the interpretation of results regarding the functional role of DLG2 in neuronal postsynaptic membranes

Additional file 4: of Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Dlg2 isoforms expressed in IFNβ-producing pDCs and cDCs. (PDF 560 kb

Additional file 2: of Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Expression of Dlg2 in B cells. (PDF 446 kb

Additional file 1: of Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Expression of Dlg2 in IFNβ/YFP-producing pDCs and cDCs. (PDF 428 kb

Additional file 3: of Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Proposed Dlg2 exon names/numbers, length, and location within the genomic sequence and accession numbers of reference sequences they are contained in. (DOCX 44 kb

Enterococcus faecalis promotes innate immune suppression and polymicrobial catheter-associated urinary tract infection

International audienceEnterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coliE. faecalis and E. coli co-infection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to E. coli infection alone. Finally, we demonstrated that co-inoculation of E. faecalis with a commensal strain of E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support that E. faecalis suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis especially when co-infected with less virulent or commensal E. coli strains

Polymersomes as Stable Nanocarriers for a Highly Immunogenic and Durable SARS-CoV-2 Spike Protein Subunit Vaccine

10.1021/acsnano.1c01243ACS NANO151015754-1577