Journal: Cell reports

Article Title: In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

doi: 10.1016/j.celrep.2020.108320

Figure Lengend Snippet: (A–C) Different subsets in the hematopoietic hierarchy from HSCs to T cell progenitors were isolated from the bone marrow and the thymus of C57BL/6 WT mice and seeded into M-ATOs. From the bone marrow: HSC (hematopoietic stem cell) (Lin − Sca1 + cKit + CD48 CD150 + IL7R − ); MPP (multi-potent progenitor) (Lin − Sca1 + c-Kit + CD48 − CD150 − IL7R − ); LMPP (lymphoid-primed multi-potent progenitor) (Lin − Sca1 + c-Kit + IL7R + Flk2 + ); and CLP (common lymphoid progenitor) (Lin − Sca1 Lo c-Kit Lo IL7R + Flk2 + ). From the thymus: ETP (Lin − CD4 − CD8 − c-Kit hi CD44 hi CD25 − ); DN2 (Lin − CD4 − CD8 − c-Kit hi CD44 hi CD25 + ); and DN3 (Lin − CD4 − CD8 − CD44 − CD25 + ). Representative phenotypes of M-ATO-derived cells are shown at weeks 1 (A), 2 (B), and 6 (C). Data are representative of three biological replicates. (D) Frequencies of T cell populations shown as percentage of total CD45 + TCRγδ − Lin − cells initiated from the different hematopoietic subsets in week 1, week 2, and week 6 M-ATOs. Error bars denote ± SD (n = 3 independent experiments).

Article Snippet: Anti-mouse CD127 (IL-7R) (Clone REA680) , Miltenyi Biotech , Cat# 130–122-938, RRID:AB_2783928.

Techniques: Isolation, Derivative Assay

Journal: Cell reports

Article Title: In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

doi: 10.1016/j.celrep.2020.108320

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD127 (IL-7R) (Clone REA680) , Miltenyi Biotech , Cat# 130–122-938, RRID:AB_2783928.

Techniques:

Journal: Cell reports

Article Title: In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

doi: 10.1016/j.celrep.2020.108320

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD127 (IL-7R) (Clone REA680) , Miltenyi Biotech , Cat# 130–122-938, RRID:AB_2783928.

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

doi: 10.1016/j.celrep.2020.108320

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse CD127 (IL-7R) (Clone REA680) , Miltenyi Biotech , Cat# 130–122-938, RRID:AB_2783928.

Techniques: Virus, Recombinant, Plasmid Preparation, Selection, Isolation, Cell Stimulation, Proliferation Assay, Activation Assay, Software

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