Journal: Science Advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: HEK293FT cells were infected with a lentivirus antibody library and individually coencapsulated with Jurkat/NF-κB-GFP-hCD40 reporter cells and fluorescence-labeled secondary antibodies in droplets. Droplets containing reporter cells activated by antibodies secreted by the coencapsulated antibody–expressing cells were sorted. The sorted cells were expanded for the second round of selection, and enriched antibodies were identified by next-generation sequencing. ( A ) Schematic of possible time traces. ( B ) Proportions of different types of droplets for each round of selection were analyzed. ( C ) Bright-field and fluorescence images of the sorted droplets after the second round of selection. ( D ) Bar plot for the top 20 scFv clusters and their frequencies during the selection process. ( E ) The change in frequencies of the selected antibodies during the selection process. ( F ) Agonist activity of the selected antibodies was determined using the CD40 reporter cell line in the presence or absence of the cross-linking secondary antibody.

Article Snippet: Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Infection, Fluorescence, Labeling, Expressing, Selection, Next-Generation Sequencing, Activity Assay

Journal: Science Advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: ( A ) The FcγRIIB dependency of antibody C04. Jurkat/NF-κB-GFP-hCD40 reporter cells were stimulated with C04 antibody or anti-HEL antibody in coculture with FcγRIIB-overexpressing HEK293FT cells. Activation of the reporter cell line was analyzed by flow cytometry. ( B ) Activation of DCs or B cells by C04. DCs or B cells isolated from a donor were stimulated by C04 in the presence (left) or absence (right) of the cross-linking secondary antibody. Expression of CD86 was analyzed by flow cytometry. ( C ) OVA-specific CD8 + T cell response induced by C04 in FcγR/CD40-humanized mice. Transgenic mice were adoptively transferred with OVA-specific OT-I cells and treated with DEC-OVA, together with C04 or isotype control antibody. Mice were euthanized for the analysis of T cells. Each circle represents an individual mouse. ( D ) Antitumor effect of C04 in the syngeneic mouse model. FcγR/CD40-humanized mice were subcutaneously engrafted with MC38 tumor cells. When MC38 tumors were established (~100 mm 3 ), mice were treated with C04, CP-870,893, or isotype control antibody. Tumor volume and body weight were measured every 3 days until the end of the experiment. Data are represented as the means ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Activation Assay, Flow Cytometry, Isolation, Expressing, Transgenic Assay, Control

Journal: Frontiers in Immunology

Article Title: Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity

doi: 10.3389/fimmu.2022.835690

Figure Lengend Snippet: AN3025 binds to human TNFR2 and cynomolgus TNFR2 selectively. (A) Binding assay of AN3025 to human TNFR2 on plate by ELISA (EC 50 = 0.052nM). (B) Binding assay of AN3025 to human TNFR1 on plate by ELISA. (C) Binding assay of AN3025 to cynomolgus TNFR2 on plate by ELISA (EC 50 = 0.053nM). (D) Binding assay of AN3025 to mouse TNFR2 on plate by ELISA. (E) Binding assay of AN3025 to rat TNFR2 on plate by ELISA. The ELISA assays were tested in duplicates. Values were expressed as Mean ± SEM.

Article Snippet: Human TNFR2 (Acro Biosystem, TN2-H5227), human TNFR1 (Acro Biosystem, TN1-H5222), cynomolgus TNFR2 (Sino Biological,90102-C08H), mouse TNFR2 (R&D systems, 426-R2-050/CF) or rat TNFR2 (R&D systems, 8348-R2-050) was coated on high binding polystyrene flat bottom micro-titer plates (Thermo Scientific, 3455).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: B cell lineage reconstitution underlies CAR-T cell therapeutic efficacy in patients with refractory myasthenia gravis

doi: 10.1038/s44321-024-00043-z

Figure Lengend Snippet: ( A ) A schematic overview of the time points at which patients receive different treatments before CAR-T infusion. # Patient MG-1 was treated with IVIG 2 g/Kg + intravenous pulse steroid 500 mg* 3days for myasthenia crisis. ( B ) A schematic overview of CAR-T treatment procedure. CAR T-cell kinetics are shown by the CAR copies per μg genomic DNA at serial time points post infusion detected by droplet digital PCR. ( C ) Representative plots showing FACS analysis stained for CAR-T cells with FITC-labeled human BCMA Fc tag protein and APC/Cy7 anti-human CD3 antibody in patient MG-1 at day 10 after CAR T-cell infusion. CAR T-cell percentage in circulating CD3 + T lymphocytes at serial time points after treatment. ( D ) Timelines of patients with cytopenia of grade 3 or higher at baseline and indicated time points after CAR T-cell infusion. BL baseline. Kinetic changes in numbers of circulating total white blood cells, neutrophils, monocytes and platelets. ( E ) Heatmap depicting protein levels of inflammatory mediators in blood following CAR T-cell infusion. Interleukin IL, TNF tumor necrosis factor, IFN interferon, CRP C-reactive protein, PCT procalcitonin. Average levels are normalized from the baseline. .

Article Snippet: Website links for the antibodies used in the flow cytometry are following: PerCP/Cyanine5.5 anti-human CD45 Antibody (Biolegend, 304028): https://www.biolegend.com/en-us/products/percp-cyanine5-5-anti-human-cd45-antibody-4240 ; FITC anti-human CD3 antibody (BD Biosciences, 561802): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd3.561802 ; PE/Cyanine7 anti-Human CD4 (BD Biosciences, 560649): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/pe-cy-7-mouse-anti-human-cd4.560649 ; APC/Cyanine7 anti-Human CD8 (BD Biosciences, 557834): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/apc-cy-7-mouse-anti-human-cd8.557834 ; APC anti-human CD19 Antibody (Biolegend, 302212): https://www.biolegend.com/en-us/products/apc-anti-human-cd19-antibody-715 ; PE anti-human CD16 Antibody (Biolegend, 302056): https://www.biolegend.com/en-us/products/pe-anti-human-cd16-antibody-569 ; PE anti-human CD56 Antibody (Biolegend, 318306): https://www.biolegend.com/en-us/products/pe-anti-human-cd56-ncam-antibody-3796 ; FITC anti-Human CD38 (BD Biosciences, 567147): https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd38.567147 ; PerCP/Cyanine5.5 anti-Human CD27 (BD Biosciences, 560612): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/percp-cy-5-5-mouse-anti-human-cd27.560612 ; PerCP anti-Human CD45 (BD Biosciences, 347464): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/clinical-discovery-research/single-color-antibodies-ruo-gmp/percp-mouse-anti-human-cd45.347464 ; APC/Cyanine7 anti-human CD3 Antibody (Biolegend, 344818): https://www.biolegend.com/en-us/products/apc-cyanine7-anti-human-cd3-antibody-6940 ; FITC-labeled human BCMA Fc tag protein (Acrobiosystems, BCA-HF254): https://www.acrobiosystems.cn/P875-FITC-Labeled_Human_BCMA_%7C_TNFRSF17_Protein_Fc_Tag.html .

Techniques: Digital PCR, Staining, Labeling

Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: Communications Biology

Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

doi: 10.1038/s42003-021-01649-6

Figure Lengend Snippet: a Illustration of antibody detection assay. Biotinylated S-RBD or Nucleocapsid proteins are captured by streptavidin-coated beads, then incubated with plasma samples and stained with PE-conjugated anti-IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4 antibodies. Fluorescence intensity analyzed by flow cytometry. b Histogram overlays demonstrating the detection of anti-S-RBD human IgG antibody (left) and soluble ACE2-Fc (right) as positive controls for plasma antibody assay. c Representative patient plasma titration. Healthy control plasma at 1:100 dilution was used as a negative control. Serial dilutions were used in the flow cytometry overlay. d Comparison of IgG antibody levels captured by S-RBD, S1 subunit of spike, S1 N terminal domain (NTD), S2 extracellular domain (ECD) and nucleocapsid protein coated beads ( n = 46 biologically independent samples). e Correlation and comparison of bead-based assay S-RBD IgG antibody levels with ELISA-based assay ( n = 44). Two-tailed Mann–Whitney U test was used to determine the statistical significance in ( d ) and two-tailed Spearman’s was used for correlation significance in ( e ). Horizontal bars in ( d ) and ( e ) indicate mean values.

Article Snippet: Wild type and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).

Techniques: Detection Assay, Incubation, Clinical Proteomics, Staining, Fluorescence, Flow Cytometry, Titration, Control, Negative Control, Comparison, Bead-based Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Communications Biology

Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

doi: 10.1038/s42003-021-01649-6

Figure Lengend Snippet: a Measurement of spike protein and nucleocapsid protein-specific IgG and spike protein-specific IgM and IgA antibodies as described in Fig. . Area under the curve (AUC) values of plasma antibodies were calculated from reciprocal dilution curves in antibody detection assay ( n = 256 for S-RBD IgG and Nucleocapsid IgG, n = 50 for S-RBD IgM, n = 144 for S-RBD IgA). Dotted lines indicate the negative threshold calculated by adding 1 standard deviation to the mean AUC values of healthy controls’ plasma. Horizontal bars show the mean value. Green, blue, salmon, red and yellow dots indicate negative controls, outpatient, hospitalized, ICU/deceased and plasma donor subjects, respectively. b S-RBD-specific IgG subclass AUC levels ( n = 144 for S-RBD IgG1, n = 74 for S-RBD IgG2, S-RBD IgG3 and S-RBD IgG4) c S-RBD IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115) d Nucleocapsid protein IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and convalescent plasma donors ( n = 115). e S-RBD IgA AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115). f S-RBD IgG AUC values of severity groups and plasma donors subdivided into males and females ( n = 115). Green dots show female subjects while purple squares indicate male subjects. Statistical significances were determined using two-tailed Mann–Whitney U test.

Article Snippet: Wild type and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).

Techniques: Clinical Proteomics, Detection Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: Communications Biology

Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

doi: 10.1038/s42003-021-01649-6

Figure Lengend Snippet: a Neutralization (NT50) of COVID-19 plasma correlated with S-RBD IgG ( n = 113), S-RBD IgA ( n = 113), S-RBD IgM ( n = 40), and nucleocapsid IgG ( n = 113). b Correlation of NT50 with S-RBD-specific IgG subclasses; IgG1 ( n = 113), IgG2 ( n = 74), IgG3 ( n = 74), and IgG4 ( n = 74). c Correlation of S-RBD IgG ( n = 115), nucleocapsid IgG ( n = 115), S-RBD IgA ( n = 115), and NT50 ( n = 113) with age. Two-tailed Spearman’s was used to determine statistical significances.

Article Snippet: Wild type and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).

Techniques: Neutralization, Clinical Proteomics, Two Tailed Test