Journal: Nature nanotechnology

Article Title: Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy

doi: 10.1038/s41565-022-01245-7

Figure Lengend Snippet: a, Schematic of the nanoparticle-based conversion strategy via anti-HER2 antibody targeting and SLAMF7 labeling of HER2-expressing cancer cells. b, Size distribution of unconjugated PEG-PLGA nanoparticles (NPs) and BiTNHER measured by dynamic light scattering. The increased size indicated the successful conjugation of antibody/protein onto the NP surface. c, Gel electrophoresis of unconjugated NP and NP conjugated with anti-HER2 antibody (HER-NP), SLAMF7 (S-NP), and both (BiTNHER). Experiment was repeated twice with similar results between repeats. d, HER-NP labeling of the HER2-expressing human breast cancer cell line SK-BR-3. (Left) Confocal images of HER2low MDA-MB-468 cells and HER2high SK-BR-3 cells after incubation with IgG-NP or HER-NP. Green, NPs; blue, DAPI (scale bar, 50 μm). (Right) FACS semiquantitative analysis of binding. e, BiTNHER labeling of HER2-expressing SK-BR-3 cells with SLAMF7. (Left) Confocal images of HER2low MDA-MB-468 cells and HER2high SK-BR-3 cells after incubation with IgG-SLAMF7-NP or HER-SLAMF7-NP. Cells were stained with anti-SLAMF7 antibody and anti-human IgG antibody (scale bar, 50 μm). Higher-magnification image of SK-BR-3 cells in the outlined area (scale bar, 20 μm). (Right) FACS semiquantitative analysis of binding. f, Binding affinity of nanoparticles with different HER:SLAMF7 conjugation ratios to HER2-expressing SK-BR-3 cells. The dissociation constant Kd increased when the conjugation ratio of anti-HER2 antibody decreased.

Article Snippet: To synthesise conjugated NPs, amine-reactive polymers were directly added into and reacted with PBS solution containing anti-HER2 antibodies (the human monoclonal anti-HER2 antibody trastuzumab from Genentech or the mouse monoclonal anti-HER2/neu antibody clone 7.16.4 from BioXcell), or recombinant SLAMF7 (human recombinant SLAMF7 from MyBioSource #MBS1458102, or mouse recombinant SLAMF7 from Genscript (Lot# U870KEL260-5)).

Techniques: Labeling, Expressing, Conjugation Assay, Nucleic Acid Electrophoresis, Incubation, Binding Assay, Staining

Journal: Nature nanotechnology

Article Title: Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy

doi: 10.1038/s41565-022-01245-7

Figure Lengend Snippet: a, BiTNHER with a 3:1 SLAMF7:HER conjugation ratio had the maximum pro-phagocytosis effect of human THP-1 against HER2-expressing SK-BR-3 cancer cells in the presence of aCD47 (n=3). b, BiTNHER converted HER2/neu-expressing human (SK-BR-3) and mouse (EO771/E2) breast cancer cells into SLAMF7high cells and promoted human THP-1 or mouse (C57BL6 bone marrow) macrophage phagocytosis in the presence of aCD47, comparable with the SLAMF7-expressing mouse leukemia L1210 cells (n=3). c, Anti-SLAMF7 antibody abrogated the pro-phagocytosis effect of BiTNHER and aCD47 on HER2-expressing cancer cells (n=3). d, Phagocytosis of CFSE-labelled HER2low EO771 and HER2high EO771/E2 mouse breast cancer cells and SLAMF7high L1210 mouse leukemia cells by mouse bone marrow macrophages in the presence of aCD47 after treatment with NP alone, NP with unconjugated anti-HER2 antibody and SLAMF7, or BiTNHER. Red, macrophages; green, cancer cells (scale bar, 50 μm). e, BiTNHER with aCD47 promotes macrophage phagocytosis against HER2-expressing breast cancer cells. f, Macrophages had increased antigen presentation of the H2kb-SIINFEKL complex after phagocytosis of BiTNHER -treated HER2-expressing EO771/E2-cOVA cells. Green, macrophages; red, H2kb-SIINFEKL complex (scale bar, 50 μm). g, Combination of BiTNHER and aCD47 increased macrophage antigen presentation of HER2-expressing cancer cells (n=4). h,i, BiTNHER with aCD47 promoted the priming of cOVA antigen-specific T cells (left) and induced a shift in naive T cells towards memory T cells (right) (n=4). For all figures, data are presented as mean±s.e.m.; **P<0.01, ***P<0.001, and ****P<0.0001 by one-way ANOVA with a Bonferroni post hoc correction. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired Student’s t-test for the indicated comparisons; n.s., not significant.

Article Snippet: To synthesise conjugated NPs, amine-reactive polymers were directly added into and reacted with PBS solution containing anti-HER2 antibodies (the human monoclonal anti-HER2 antibody trastuzumab from Genentech or the mouse monoclonal anti-HER2/neu antibody clone 7.16.4 from BioXcell), or recombinant SLAMF7 (human recombinant SLAMF7 from MyBioSource #MBS1458102, or mouse recombinant SLAMF7 from Genscript (Lot# U870KEL260-5)).

Techniques: Conjugation Assay, Expressing

Journal: Cell metabolism

Article Title: GDF15 Induces Anorexia through Nausea and Emesis

doi: 10.1016/j.cmet.2019.12.004

Figure Lengend Snippet: (A) Hepatic GDF15 mRNA was higher at day 1 after cisplatin injection compared to controls. No significant differences in GDF15 mRNA expression occurred at day 3 and day 7.

Article Snippet: Mouse beta-actin (Mm00607939, lot P180325–001) was used as housekeeping reference gene and the following probes were used to amplify GDF15 (Mm00442228, lot 1670789), TNF-α (Mm00443258, lot 1629748), and COX-2 (Mm03294838, lot 1635964) transcripts.

Techniques: Injection, Expressing

Journal: Cell metabolism

Article Title: GDF15 Induces Anorexia through Nausea and Emesis

doi: 10.1016/j.cmet.2019.12.004

Figure Lengend Snippet: (A) The preference for the paired solution was significantly reduced after centrally delivered GDF15 (30 pmol i.c.v., n = 7) compared to negative controls (DMSO, 1 µL i.c.v., n = 4). LiCl (0.15 M, i.p., n = 6) was used as a positive control.

Article Snippet: Mouse beta-actin (Mm00607939, lot P180325–001) was used as housekeeping reference gene and the following probes were used to amplify GDF15 (Mm00442228, lot 1670789), TNF-α (Mm00443258, lot 1629748), and COX-2 (Mm03294838, lot 1635964) transcripts.

Techniques: Positive Control

Journal: Cell metabolism

Article Title: GDF15 Induces Anorexia through Nausea and Emesis

doi: 10.1016/j.cmet.2019.12.004

Figure Lengend Snippet: (A–C) Central administration of GDF15 (10 and 30 pmol into the 4th ventricle) dose-dependently induced anorexia (A), kaolin consumption (B), and body weight loss (C) in obese rats (n = 14) compared to vehicle treated animals.

Article Snippet: Mouse beta-actin (Mm00607939, lot P180325–001) was used as housekeeping reference gene and the following probes were used to amplify GDF15 (Mm00442228, lot 1670789), TNF-α (Mm00443258, lot 1629748), and COX-2 (Mm03294838, lot 1635964) transcripts.

Techniques:

Journal: Cell metabolism

Article Title: GDF15 Induces Anorexia through Nausea and Emesis

doi: 10.1016/j.cmet.2019.12.004

Figure Lengend Snippet: (A) In seven of the eight animals tested, 1mg/kg of GDF15 induced robust emetic responses that were not observed after 0.1 mg/kg GDF15 or saline injections.

Article Snippet: Mouse beta-actin (Mm00607939, lot P180325–001) was used as housekeeping reference gene and the following probes were used to amplify GDF15 (Mm00442228, lot 1670789), TNF-α (Mm00443258, lot 1629748), and COX-2 (Mm03294838, lot 1635964) transcripts.

Techniques: Saline

Journal: Cell metabolism

Article Title: GDF15 Induces Anorexia through Nausea and Emesis

doi: 10.1016/j.cmet.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse beta-actin (Mm00607939, lot P180325–001) was used as housekeeping reference gene and the following probes were used to amplify GDF15 (Mm00442228, lot 1670789), TNF-α (Mm00443258, lot 1629748), and COX-2 (Mm03294838, lot 1635964) transcripts.

Techniques: Recombinant, RNA Extraction, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Reports

Article Title: Identification of presented SARS-CoV-2 HLA class I and HLA class II peptides using HLA peptidomics

doi: 10.1016/j.celrep.2021.109305

Figure Lengend Snippet:

Article Snippet: Cells were stained with mouse monoclonal IgG1 anti-ACE2 antibody (cat: sc-390851, clone E-11, lot: D2420, Santa Cruz) or anti-TMPRSS2 (cat: sc-515727, clone:H-4, lot:D2420), followed by Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (cat: 115-545-146, lot: 138610).

Techniques: Purification, Staining, Virus, Plasmid Preparation, Recombinant, Software

Journal: Neuron

Article Title: Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition

doi: 10.1016/j.neuron.2022.04.017

Figure Lengend Snippet:

Article Snippet: The following antibodies were used in this study: rabbit polyclonal anti-β-actin (Abcam; Cat# ab8227; RRID: AB_2305186; LOT# GR3314266-1), mouse monoclonal anti-calbindin (Swant; Cat# 300; RRID: AB_10000347; LOT# 17 (F)), goat polyclonal anti-calretinin (Swant; Cat# CG1; RRID: AB_10000342; LOT# 1§.1), mouse monoclonal anti-CamKII alpha (Thermo Fisher Scientific; 6G9; Cat# Ma1-048; RRID: AB_325403; LOT# TH269517), mouse monoclonal anti-cannabinoid receptor 1 (Immunogene; IMG-3C2; Cat# IMG-CB1R-mAb001; LOT# CJ03), guinea pig polyclonal anti-cholecystokinin (Synaptic Systems, Cat# 438004; RRID: AB_2814938; LOT# 1-1), mouse monoclonal anti-GAD67 (Millipore; 1G10.2; Cat# MAB5406; RRID: AB_2278725; LOT# 3015328), rabbit polyclonal anti-GAPDH (Enogene; Cat# E1C604; LOT# R14Q12), mouse monoclonal anti-gephyrin (Synaptic Systems; mAb7a; Cat# 147021; RRID: AB_2232546; LOT# 147021/15), mouse monoclonal anti-gephyrin (Synaptic Systems; mAb7a; Cat# 147011; RRID: AB_887717; LOT# 147011/54), rat monoclonal anti-HA (Roche; 3F10; Cat# 11867431001; RRID: AB_390919; LOT# 34502100), rabbit monoclonal anti-HA (Cell Signaling; 3724; Cat# 3724; RRID: AB_1549585; LOT# 9), guinea pig polyclonal anti-MAP2 (Synaptic Systems; Cat# 188004; RRID: AB_2138181; LOT# 2-26), mouse monoclonal anti-MAP2 (Synaptic Systems; 198A5; Cat# 188011; RRID: AB_2147096; LOT# 1-10), rabbit polyclonal anti-neurexin , chicken anti-neurexin , rabbit anti-neuroligin , rabbit monoclonal anti-nNOS (Cell Signaling; C7D7; Cat# 4231; RRID: AB_2152485; LOT# 2), goat polyclonal anti-parvalbumin (Swant, Cat# PVG214; RRID: AB_10000345), mouse monoclonal anti-PSD95 (Santa Cruz; 7E3; Cat# sc32290; RRID: AB_628114; LOT# J1509), goat polyclonal anti-somatostatin (Santa Cruz; Cat# sc7819; RRID: AB_2302603; LOT# L1611), mouse monoclonal anti-Synaptotagmin 2 (Zebrafish International Resource Center; Cat# znp-1; RRID: AB_10013783), mouse monoclonal anti-V5 (Biorad; SV5-PK1; Cat# MCA1360; RRID: AB_322378; LOT# 148239), guinea pig polyclonal anti-vGAT (Synaptic Systems; Cat# 131004; RRID: AB_887873; LOT# 2-42), guinea pig polyclonal anti-vGlut1 (Millipore; Cat# AB5905; RRID: AB_2301751; LOT# 3308226), rabbit polyclonal anti-VIP (Immunostar; Cat# 20077; RRID: AB_572270; LOT# 1513001), guinea pig anti-somatostatin (raised in the present study against amino acid residues 35-88 of mouse pro-somatostatin, GenBank: #BC010770.1), goat anti-cannabinoid receptor 1 (Nittobo Medical, MSFR100600; RRID: AB_2571592), guinea pig anti-GABAA receptor alpha 1 (Nittobo Medical, MSFR101540; RRID: AB_2571572), goat anti-vGAT (Nittobo Medical, MSFR106130; RRID: AB_2571623), guinea pig anti-PSD95 (Nittobo Medical, MSFR105180; RRID: AB_2571612).

Techniques: Virus, Recombinant, Reporter Assay, RNAscope, Multiplex Assay, Plasmid Preparation, Software, Targeted Proteomics, Microscopy

Journal: Cells

Article Title: IFN-γ Induces PD-L1 Expression in Primed Human Basophils

doi: 10.3390/cells11050801

Figure Lengend Snippet: The effect of TSLP on the expression of PD-L1 induced by IFN-γ in primary human basophils. Basophils (0.1 × 10 6 cells/200 μL/96-well plate) isolated from PBMCs of healthy donors were cultured with either IL-3, TSLP (10 ng/mL), TSLP + IL-3 or TSLP + IFN-γ. Basophil phenotype was evaluated by flow cytometry after 24 h. Expression of FcεRI, CD69, CD107a, PD-L1, and PD-L2 on the basophils (% positive cells and median fluorescence intensities (MFI), mean ± SD; n = 3 independent donors from three independent experiments) was presented. ns, not significant, * p < 0.05, one-way ANOVA Friedman test with Dunn’s multiple comparisons post-test.

Article Snippet: ImmunoTools (Friesoythe, Germany): recombinant human IL-3 (Catalogue: 11,340,037) and recombinant IFN-γ (Catalogue: 11,343,534, lot specific activity: 2 × 10 7 IU/mg); R&D systems: recombinant thymic stromal lymphopoietin (TSLP) (Catalogue: 1398-TS, lot: IDK0721051); Miltenyi Biotec: IFN-γ (Catalogue: 130-096-484, lot specific activity: ≥2 × 10 7 IU/mg, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Catalogue: 130-093-867, lot specific activity: 1.6 × 10 7 IU/mg); Sigma-Aldrich (St. Quentin Fallavier, France): Anti-IgE antibodies (Catalogue: I6284, lot: SLCF0672), Lipopolysaccharide (LPS: E. coli 055:B5); eBioscience (Paris, France): Fixable viability dye eFluor 506 (1:500 dilution); Lonza (Verviers, Belgium): Serum-free X-VIVO 15 medium.

Techniques: Expressing, Isolation, Cell Culture, Flow Cytometry, Fluorescence

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Blended projections of T cells (red) clustered around a conduit (white)-associated VACV-infected cell (green) near a high endothelial venule (HEV, indicated in picture). Arrows point to conduits. Scalebar = 20 μm. Area in circle is magnified on the right. Scalebar = 5 μm. ERTR7= white, VACV-infected cells = green, OT-I CD8 + T cells = red, B220 = blue. Results are representative of 30 LN sections taken from 5 experiments. b ) OT-I CD8 + T cell activation as determined by flow cytometry of single-cell suspensions of popliteal LNs harvested 8 h p.i. 10 6 OT-I cells were transferred 12–24 h prior to infection. NP-eGFP (no SIINFEKL) was given at 10 7 pfu; all other infections used NP-S-eGFP (containing SIINFEKL) at the indicated dose. Activation was determined by CD69 expression. MFI is shown on the right. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. c ) OT-I CD8 + T cell activation in (b) but 24 h p.i. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. d ) Quantitation of the percentage of VACV- or MVA-infected cells in either the SCS and IFA region or T cell zone contacted by OT-I CD8 + T cells (n = 37 VACV or 32 MVA; dots indicate individual sections). Bars = mean. Error bars = SEM. e ) Percentage of activated OT-I CD8 + T cells (those with CD69 MFI > 50) in each region of 10 LN sections harvested 8 h after infection with 10 8 pfu VACV. Percentages shown are activated cells/total cells in each region. Dots = individual LN sections. Bars = mean. Error bars = SEM. f ) Percentage of only the activated OT-I CD8 + T cells (as opposed to total cells in (e)) found in each LN region. g ) MIP LN section 8 h p.i. showing OT-I CD8 + T cell activation. B = B cell follicle, SCS = subcapsular sinus. Arrow indicates activated T cell cluster in the T cell zone, magnified on the right. Scalebars = 50 μm, left and 5 μm, right. OT-I CD8 + T cells = red, CD69 = white, VACV-infected cells = green, B220 = blue. h ) MFI of CD69 on all activated cells in each region of the popliteal LN shown in (g). n =197 T cells. Bars = mean. Error bars = SEM. i ) MIP of popliteal LN harvested 8 h post infection with 10 8 pfu VACV (green) without OT-I transfer. Endogenous, polyclonal CD8 + T cells = red. Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. B220 = blue, VACV-infected cells = green. The boxed area is magnified in (j). scalebars = 100 μm j ) Higher magnification MIP image of (i) showing both the SCS&IFA (labeled SCS) and T cell zone (T). Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. scalebars = 50 μm. f,h,i,j) 6 LNs from 3 experiments were analyzed. g) 10 LNs from 5 experiments were analyzed. Statistics = unpaired two-tailed t test.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Infection, Activation Assay, Flow Cytometry, Expressing, Quantitation Assay, Labeling, Two Tailed Test

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Maximum intensity projection (MIPs) of sections from two different popliteal LNs (left and right) harvested 8 h after footpad injection of 10 6 pfu of VACV. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue. Arrows indicate the location of VACV-infected paracortical cell. scalebars = 100 μm. Higher magnification images are shown on the right; lower panels lack the blue channel for clarity. Scalebars = 50 μm. Results are representative of 10 LNs from 3 experiments. b ) MIP of a section of a popliteal LN harvested 8 h after footpad injection of 10 6 pfu of VACV. Prior to infection, 10 6 OT-I CD8 + T cells were transferred (pink). ERTR7 = red, B220 = blue, CD69 = white, VACV-infected cells = green. Arrows indicate two areas of T cell activation that are shown in higher magnification insets on the left. Scalebar = 200 μm. Top panels of insets show merge; bottom panels do not show OT-I signal (pink) in order to reveal CD69 staining more clearly. Scalebars = 20 μm. Results are representative of 10 LNs from 3 experiments. c ) MIP of a section of a popliteal LN harvested 8 h after footpad infection with 10 6 pfu of VACV showing an infected paracortical CD205 + DC. Scalebar = 200 μm. Higher magnification images are shown in the insets showing: top left) merge; top right) conduits + VACV-infected cells; lower left) conduits + VACV-infected cells + CD11c + CD11b; lower right) conduits + VACV-infected cells + CD205. Scalebar = 20 μm. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue, CD11c = yellow, CD205 = purple, and CD11b = brown. Results are representative of 10 LNs from 3 experiments.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Injection, Infection, Activation Assay, Staining