Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: IL-23R and IL-12R expression in ex vivo CLL cells assessed by flow cytometry. FSC is proportional to cell size and SSC reflects intracellular complexity/granularity (each dot represents one cell). Axes in marker plots show fluorescence intensity for the indicated markers. Percentages within gates indicate the fraction of cells relative to the parent population. Lymphocytes were identified by FSC/SSC properties, followed by doublet exclusion using pulse height vs. width plots. Within the single-cell gate, CD19 + /CD5 + CLL cells were selected. This population was further sub-divided into RF and PF based on the differential expression of CD184 and CD5. ( A ) Gating strategy used to identify lymphocytes, single cells, CD19 + CLL cells, and to discriminate proliferative fraction (PF) and resting fraction (RF); ( B ) surface expression of IL-23R, IL-12Rβ1, and IL-12Rβ2 receptor subunits in ex vivo CLL cells, shown for CD19 + PF and RF populations; ( C ) representative flow cytometry plots showing sorting of PF and RF populations from six CLL samples according to the gates indicated; ( D ) IL-12Rβ1 mRNA expression in sorted RF and PF cells, measured by RT-qPCR. Data are shown as mean ± SEM. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. * p < 0.05; ** p < 0.01, ns: not significant. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488; PE VIO 770: near-infrared fluorochrome emitting in the ~770–780 nm range.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Expressing, Ex Vivo, Flow Cytometry, Marker, Fluorescence, Single Cell, Quantitative Proteomics, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: IL-23R and IL-12R expression in CLL cells after incubation with CpG and CpG + IL-15 for 72 h assessed by flow cytometry analyses. ( A ) Representative flow cytometry gating strategy used to identify CLL cells and to assess IL-23R and IL-12R complex expression under the indicated culture conditions (medium alone, CpG, or CpG + IL-15) after 72 h; ( B ) percentage of CLL cells expressing the IL-23R receptor subunit, IL-12Rβ1 receptor subunit, and IL-12Rβ2 receptor subunit following 72 h incubation under the indicated conditions; ( C ) percentage of CLL cells expressing the IL-23R complex and the IL-12R complex following 72 h incubation under the indicated conditions. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. * p < 0.05; **** p < 0.0001. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Expressing, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: Differential expression of IL-23R and IL-12R in CLL RF and PF subsets after stimulation with CpG and CpG + IL-15 assessed by flow cytometry analyses. ( A ) Representative flow cytometry gating strategy used to identify CLL cells and to distinguish RF and PF subsets, followed by assessment of IL-23R and IL-12R complex expression after 72 h incubation with CpG or CpG + IL-15; ( B ) percentage of RF and PF CLL cells expressing the IL-23R receptor subunit, IL-12Rβ1 receptor subunit, and IL-12Rβ2 receptor subunit after 72 h incubation under the indicated conditions; ( C ) percentage of RF and PF CLL cells expressing the IL-23R complex and the IL-12R complex after 72 h incubation under the indicated conditions. Each symbol represents an individual CLL sample; horizontal bars indicate mean ± SEM. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. ** p < 0.01; **** p < 0.0001, ns: not significant. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488; BV421 = Brilliant Violet 421.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Quantitative Proteomics, Flow Cytometry, Expressing, Incubation

Journal: PloS one

Article Title: Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

doi: 10.1371/journal.pone.0006027

Figure Lengend Snippet: Figure 1. Circulating IL-6 and muscle IL-6 mRNA and IL-6 receptor response muscle lengthening contractions (MLC). (1a) Average serum interleukin-6 (IL-6) concentration. Time 0 hr corresponds to pre-intervention values; all other time-points correspond to post-intervention time (hr). (1b) Relative IL-6 mRNA expression, expressed as fold-change from 0 hr. (1c) Pearson correlation of serum IL-6 concentration versus muscle IL-6 mRNA (fold-change), correlation is representative of the individual data points and presented as mean values ($)6SD (error bars). (1d) Relative IL-6 receptor (IL-6Ra) mRNA expression, expressed as fold-change from 0 hr. (1e) Immunofluorescent image (406) of a muscle cross-section triple-stained for Pax7 (red), IL-6Ra (green) and nuclei (DAPI = blue); IL-6Ra staining is apparent on the sarcolemma and satellite cell membrane. White arrow denotes one of the Pax7+ nuclei which co-localized with IL-6Ra (scale bar = 100 mm). Values are reported as mean6S.E.M. Mean values represent the mean for all 8 subjects per time-point (8 samples per time-point, 40 samples total). *p,0.05 vs. 0 hr. doi:10.1371/journal.pone.0006027.g001

Article Snippet: Immunofluorescence 7 mm sections were cryosectioned and stained with antibodies against Pax7 (neat; DSHB, USA); IL-6 (500 ng/mL, MAB 2061, R&D Systems, USA); p-STAT3 (p-STAT3 Y705 1:100, Cell Signaling Technologies Inc., USA); IL-6Ra (1:50, MCA822, Serotec, UK); PCNA (1:200, ab15497, Abcam Inc., USA); and Laminin (1:1000, L8271, Sigma-Aldrich, Canada).

Techniques: Concentration Assay, Expressing, Staining, Membrane

Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A and B ) Normalized cell count of classical macrophages (CD11b + CD11c – F4/80 + Gr1 – ), hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ), cDC1s (CD11b – CD11c + F4/80 – Gr1 – ), and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) per islet ( A ) or per pancreatic lymph node ( B ) from euglycemic 14-week-old NOD βST or littermate mice. ( C ) Representative flow cytometry histogram of IL-12p35 expression in cDC2s from islets of euglycemic 14-week-old NOD βST and NOD littermate mice, compared with isotype control. ( D ) Representative flow cytometry histogram of IL-12p35 expression in hybrid macrophages from islets of euglycemic NOD βST and littermate mice, compared with isotype control. Vertical line denotes the positive gate. ( E ) Quantification of IL-12p35 expression shown in C and D across 7 NOD βST mice, 16 NOD littermate mice, and 5 isotype controls. Error bars represent SD. Mann-Whitney U tests were used to examine statistical differences between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Cell Counting, Flow Cytometry, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A ) Confirmation of ST8Sia6-myc shutoff after 5 weeks of doxycycline treatment by IHC. Schematic of doxycycline treatment for temporal control of ST8Sia6 expression. Scale bars: 100 μm. ( B ) Diabetes-free incidence after treatment of 15 euglycemic NOD βST and 29 euglycemic littermate mice with doxycycline starting at 8 weeks of age. ( C ) Comparison with disease kinetics of 8-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 151) mice (subset from ). ( D ) Diabetes-free incidence after treatment of 12 euglycemic NOD βST and 16 euglycemic littermate mice with doxycycline starting at 20 weeks of age. ( E ) Comparison with disease kinetics of 20-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 115) mice (subset from ). Log-rank Mantel-Cox test was used to analyze statistical differences in diabetes-free incidence of indicated groups. ( F ) Insulitis distribution in pancreas sections of NOD βST mice treated with doxycycline for 5 weeks from 8 weeks or 20 weeks of age, compared with 300-day-old pancreata from NOD βST mice never treated with doxycycline (subset from ). Scoring and analysis as in . n = 69 islets from 12 mice (8-week doxycycline) or 127 islets from 11 mice (20-week doxycycline). ( G and H ) Ratio of T-bet + Tregs (T-bet + FoxP3 + CD4 + ) to Th1 T cells (T-bet + FoxP3 – CD4 + ) ( G ) or to SLECs (T-bet + CD8 + ) ( H ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. ( I ) Quantification of IL-12p35 expression in hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ) and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. Same gating schemes as in and 5. Error bars represent SD. Mann-Whitney U test was used for statistical analysis between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Control, Expressing, Comparison, MANN-WHITNEY

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: Cell reports

Article Title: High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status

doi: 10.1016/j.celrep.2021.108974

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Human/Mouse IL-5 (TRFK5)-143Nd , Fluidigm , Cat# 3143003B.

Techniques: Purification, Control, Antibody Labeling, Flow Cytometry, Software, Staining, Blocking Assay