Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: Peripheral blood activated B cells (CD27 high CD38 high ) were isolated and sorted by FACS for single cell sequencing. a) Percentage of CD27 high CD38 high cells among live B cells. Each sample is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. b) UMAP representation of 72277 CD27 high CD38 high sorted B cells from 9 COVID-19 ICU patients and 3 healthy controls. Between 2416 and 11229 cells were recovered per sample. Transcriptionally similar clusters were identified using shared nearest neighbor (SNN) modularity optimization (left). The combination of CD19, MS4A1, CD27, CD38, IFIT1, MIKI67, IRF4 and PRDM1 expression were used for annotation of the different activation/differentiation stages (right). c) Heatmap of genes that resemble markers for the six different clusters. Depicted are genes with a p-value < 0.01 (Wilcoxon rank sum test) after bonferroni correction, and an average absolute fold-change > log2(1.3). Shown are z-scores of the average expression. d) UMAP representation of the expression levels of selected signature genes for activated/differentiated B cells. e) UMAP representation of analyzed cells from one healthy control and two ICU patients, representing early (first week, patient #1) and late (>7days, patient #5) phase after ICU admission. f) Percentage of cells belonging to a defined cluster among all sequenced B cells per donor. Each dot represents one time point from one single donor. Donors were grouped as “HC” for healthy controls (n = 3), “1st week” for patients within 7 days after ICU admission (n = 6) and “Late” for patients who have been admitted to the ICU for more than a week at the time of analysis (n = 8). The line indicates the median. Significance was determined by using a two-sided analysis of varience (ANOVA) folowed by Tukey’s multiple comparison test with corrected p values as indicated in the figure.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Isolation, Sequencing, MANN-WHITNEY, Expressing, Activation Assay, Control, Comparison

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: a) 5-marker MELC panel of SARS-CoV2-positive and control lungs (SARS-CoV2-negative). Respective patient characteristics are given in supplementary Fig. 4a. Each image of each patient depicts the same field of view of the same section, sequentially stained with the fluorescence-labelled antibodies indicated and the nuclear stain DAPI. Magenta arrows indicate IgA2+IgA+CD27 + CD38 + cells (containing a nucleus). Images contain 2048 × 2048 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Scale bar: 100 µm. b) Absolute numbers of IgA2+IgA+CD27 + CD38 + cells per field of view in all MELC runs acquired (two runs per patient except for COVID-19_A with four runs and Control_B with one single run; see and supplementary Fig. 4b). Each field of view is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. c) Region of interest of one exemplary control and COVID-19 lung (as in a)), showing an overlay of the indicated markers. White arrows point out IgA2+CD27 + CD38 + cells. Scale bar: 20 µm. (d-e) Bronchoalveolar lavage (BAL) cells for single cell sequencing were enriched for CD45 + cells via MACS and live cells were further sorted using FACS (see supplementary Fig. 4c). d) UMAP of 5459 cells representing clusters containing T cells, B cells and CD14 -expressing cells from patient #1 on day 59 following ICU admission. B and T cell cluster identification based on BCR/TCR, CD19, CD3E, CD4 and CD8A expression (see supplementary Fig. 4d). UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG . e) UMAP coordinates and clustering was computed for 433 and 2723 cells from patient #9 on days 31 and 46 following ICU admission, respectively. UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG at the two different time points is shown side by side.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Marker, Control, Staining, Fluorescence, Generated, Microscopy, MANN-WHITNEY, Sequencing, Expressing

Journal: eLife

Article Title: Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case–control study

doi: 10.7554/eLife.70393

Figure Lengend Snippet:

Article Snippet: Antibody , anti-IL-21, clone 3A3-N2 (mouse monoclonal) , Miltenyi Biotec , Cat# 130-120-702 , Fluorochrome: APC.

Techniques: Staining, Concentration Assay, Recombinant, Produced, Sequencing, Isolation, Gel Extraction, Software, Adhesive, Reverse Transcription, Magnetic Beads

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 are involved in the permanently induced NF-κB activity present in thymocytes during the coculture with TEC. Thymocytes were freshly isolated (lanes 1 and 2) or cultured for 45 h (lanes 3 to 11) either alone (lane 3 [Control thymocytes]) or in the presence of TEC under conditions avoiding contact (lanes 4 to 9) or allowing contact (lanes 10 and 11). During the coculture, the cells were either left untreated (lane 4 [Control coculture, 45 hours] and lane 10 [Control coculture]) or were treated at the start of the coculture with antibodies respectively raised against TNF (lane 5), IL-6 (lane 6), and IL-1β (lane 8), with the antagonist of the IL-1 receptor (lane 7), or with αTNF plus IL-1 ra plus αIL-6 (lanes 9 and 11). Whole-cell extracts from these various samples were incubated with a 32P-labeled oligonucleotide representing the HIV LTR-derived κB motif. Competition (comp [lane 1]) with a 40-fold molar excess of unlabeled oligonucleotide was used to confirm the specificity of the DNA-binding activity detected. NF-κB activity is indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. This experiment is representative of three independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling, Derivative Assay, Binding Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 induce NF-κB activity in thymocytes, and IL-7 is required for this effect. Whole-cell extracts were prepared from freshly isolated thymocytes (panel A, lane 1) or from thymocytes cultivated for 30, 60, or 45 h. (A) During the indicated culture times, thymocytes were either left untreated (lane 1, control for freshly isolated; lane 2, control 30 h; and lane 5, control 60 h) or were stimulated with either TNF (lanes 3 and 6), IL-1β (lanes 4 and 7), or TNF in the presence of TEC CM (TEC CM + TNF) (lane 9). TEC conditioned medium was also added alone (lane 8 [TEC CM]). The data shown here are representative of three independent experiments carried out on three thymuses. (B) Thymocytes were cultured for 45 h either untreated (lane 1, control 45 h), with TNF (lane 2), with TEC CM (lane 3), or with TNF in the presence of TEC CM (TEC CM + TNF) preincubated (lane 5) or not (lane 4) with antibody against IL-7. In lanes 6 to 8, the thymocytes were left untreated for 6 h (lane 6) or incubated with an antibody against IL-7Rα (lane 7) or with an IgG1 control serum (lane 8) before being cocultured with TEC for 45 h. The data shown here are representative of two independent experiments carried out on two thymuses. (C) Cells were left untreated for the 45 h of the culture (lane 1, control 45 h) or treated with IL-1β (lane 2), TNF (lane 3), IL-1β plus TNF plus IL-6 plus GM-CSF (lane 4), IL-7 (lane 5), IL-7 plus IL-1β (lane 6), or IL-7 plus TNF (lane 7). This experiment is representative of three independent experiments carried out on three thymuses. (D) Thymocytes were left untreated (lane 1, control) or stimulated with IL-7 (lane 2) or IL-7 in presence of anti-TNF and Il-1ra (lane 3). Whole-cell extracts were incubated with a 32P-labeled oligonucleotide representing κB motif. NF-κB activity is indicated. This experiment is representative of two independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling