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: 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: Cancer Immunology Research

Article Title: Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

doi: 10.1158/2326-6066.cir-23-0127

Figure Lengend Snippet: Figure 7. Proposed mechanism of action of CARG-2020. Top, control no treatment ovarian tumors are “cold” and express/secrete IL17 and PD-L1 leading to differentiation of protumor M2 macrophages, which inhibit the expansion of cytotoxic CD8þ T cells; bottom, CARG-2020, by expressing shRNA for PD-L1 and dominant negative for IL17, in addition to promoting the secretion of IL12, shifts the environment to a “hot” tumor and promotes the differentiation of antigen-presenting cells and consequently the expansion of cytotoxic CD8þ T cells.

Article Snippet: After blocking with 5%milk, membranes were probed with the following primary antibodies at 4 C overnight: ß-Actin (Millipore, #MAB1501); IL12 (R&D Systems, #MAB1570-500); HA tag (for IL17RA; Invitrogen, #71-5500); PD-L1 (Bio X Cell, #BE0101).

Techniques: Control, Expressing, shRNA, Dominant Negative Mutation

Journal:

Article Title: Computational Design and Application of Endogenous Promoters for Transcriptionally Targeted Gene Therapy for Rheumatoid Arthritis

doi: 10.1038/mt.2009.182

Figure Lengend Snippet: Comparison of therapeutic efficacy using Saa3 versus IL1E/IL6P promoter for disease-regulated Il1rn expression. (a) Promoter activity in transduced synovium of naive and arthritic C57Bl/6 mice. Knee joints were injected with 300 ng p24gag equivalent lentivirus encoding PGK, Saa3, or IL1E/IL6P-luciferase. Seven days after transduction, arthritis was induced by intra-articular injection of 180 µg zymosan A. After 24 hours, luciferase activity was assessed ex vivo. Data are represented as individual relative luciferase activities; horizontal bars indicate the means per group. (b) Efficacy of transcriptionally targeted adenoviral vectors expressing Il1rn. NIH-3T3-5xNF-κB-Luc were transduced at a multiplicity of infection (MOI) of 10 with control vector (del) or adenovirus encoding CMV, Saa3, and IL1E/IL6P-driven Il1rn. After 24 hours, cells were transduced at an MOI of 10 with control vector (del) or Ad5.CMV-Il1b. The day thereafter, IL-1β-induced NF-κB activation was assessed by luciferase assay. Data are represented as relative luciferase activities ± SEM (n = 4), and differences were determined using analysis of variance with Dunnett's post-test. **P < 0.01.

Article Snippet: White high-binding flat bottom 96-well plates (Greiner Bio-One, Alphen a/d Rijn, the Netherlands) were coated with the capture antibody rat anti-murine Il1rn (MAB480; R&D Systems, Minneapolis, MN) at 3 μg/ml in 0.1 mol/l carbonate buffer pH 9.6 and incubated overnight at 4 °C.

Techniques: Comparison, Drug discovery, Expressing, Activity Assay, Injection, Luciferase, Transduction, Ex Vivo, Infection, Control, Plasmid Preparation, Activation Assay

Journal: Immunity

Article Title: Differentiation and protective capacity of virus-specific CD8 + T cells suggest murine norovirus persistence in an immune-privileged enteric niche

doi: 10.1016/j.immuni.2017.09.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dead cells were removed by gating on a LIVE/DEAD Aqua kit (Invitrogen, Carlsbad, CA) versus forward scatter (FSC-H). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD8β-PerCP-Cy5.5 (clone YTS156.7.7) Biolegend Cat# 126609 Anti PD-1-PE-Cy7 (clone RMP1-30) Biolegend Cat# 109109 Anti CD103-PacificBlue (clone 2E7) Biolegend Cat# 121417 Anti CD44-BV785 (clone IM7) Biolegend Cat# 103041 Anti CD45.2-BV785 (clone 104) Biolegend Cat# 109839 Anti CXCR4-BV421(clone L276F12) Biolegend Cat# 146511 Anti Ly6c-BV785 (clone HK1.4) Biolegend Cat# 128041 Anti KLRG-1-BV605 (clone 2F1) Biolegend Cat# 138419 Anti CD62L-BV605 (clone MEI-14) Biolegend Cat# 104438 Anti TNFa-PacificBlue (clone MP6-XT22) Biolegend Cat# 506318 Anti CD107a-AF488 (clone 1D4B) Biolegend Cat# 121607 Anti CD28-PE-Cy7 (clone 37.51) Biolegend Cat# 102126 Anti CX3CR1-PE (clone SA011F11) Biolegend Cat# 149006 Anti CD11a-PE (clone 2D7) Biolegend Cat# 101008 Anti FOXP3-PE (clone MF-14) Biolegend Cat# 126404 Anti CD90.1 (Thy-1.1)-BV605 (clone OX-7) Biolegend Cat# 202537 Anti CD25-PE-Cy5 (clone PC61) Biolegend Cat# 102010 Anti CD8a-PE-eF610 (clone 53.6) eBioscience Cat# 61-0081-80 Anti CD69-PE-Cy7 (clone H1.2F3) eBioscience Cat# 25-0691-82 Anti 2B4-FITC (clone eBio244F4) eBioscience Cat# 11-2441-82 Anti CD49b-PE-Cy5 (clone DX5) eBioscience Cat# 15-5971-82 Anti CD49d-FITC (clone R1-2) eBioscience Cat# 11-0492-81 Anti CD90.2 (Thy1.2)-FITC (clone 30-H12) eBioscience Cat# 11-0903-82 Anti CD27-APC-eFluor 780 (clone LG.7F9) eBioscience Cat# 47-0271-82 Anti CD45.1-APC-R700 (clone A20) BD Biosciences Cat# 565814 Anti IFN -AlexaFluor700 (clone XMG1.2) BD Biosciences Cat# 557998 Anti GITR-FITC (clone DTA-1) BD Biosciences Cat# 558139 Anti Granzyme-B-PE (clone GB11) Invitrogen Cat# GRB04 Anti MIP-1a-APC (clone 39624) R&D Systems Cat# 1C450A Anti IL-12Rβ2-PRCP (clone 305719) R&D Systems Cat# FAB1959C Anti-CD8β (Lyt 3.2) (clone 53-5.8) Bio X Cell Cat # BE0223 Anti-Thy1.1 (clone 19E12) Bio X Cell Cat # BE0214 Bacterial and Virus Strains MNV-CW3 Tomov et al., 2013 GenBank EF014462 MNV-CR6 Tomov et al., 2013 GenBank EU004676 MNV-CW3 Y→F This study N/A MNV-CR6 F→Y This study N/A rVV 519Y This study N/A Biological Samples N/A N/A N/A Chemicals, Peptides, and Recombinant Proteins Deoxyribonuclease I from Bovine Pancreas Sigma-Aldrich Cat # D5025 Collagenase/Dispase Roche Cat # 11097113001 DTT (dithiothreitol) Thermo Fisher Cat # R0861 RPMI 1640 Corning Cat # 15-040-CM GemCell U.S.

Techniques: Virus, Recombinant, Saline, Reverse Transcription, Gene Expression, SYBR Green Assay, Isolation, Staining, Microarray, Plasmid Preparation, Software