Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 1 | Engineering and characterization of receptor-based Axl antagonists. (a) Axl’s extracellular domain consists of two Ig-like domains containing high- and low-affinity Gas6 binding sites, followed by two fibronectin type III domains. Binding of Gas6 to Axl leads to receptor dimerization and activation of downstream signaling. Axl decoy receptors sequester Gas6, preventing activation of the Axl signaling cascade. (b) Overlaid flow cytometry dot plots representing binding of yeast-displayed wild-type Axl Ig1 (red) and unsorted Axl Ig1 library (blue) to 10 nM Gas6 (y axis) and expression levels on the yeast cell surface (x axis). (c) Flow cytometry histograms of the initial Axl library and intermediate sort products compared to wild-type Axl Ig1 (gray), measuring binding to 0.5 nM Gas6 (top row) and persistent Gas6 binding after a 30-h incubation with excess competitor (bottom row). MYD1 is also included for comparison. For clarity, only the gated population of yeast expressing Axl is shown. AU, arbitrary units. (d) Binding affinities of wild-type Axl Ig1, MYD1 and Axlnb to Gas6 as determined by KinExA. (e) Binding affinities to Gas6 of every permutation of the four mutations found in MYD1. Raw KinExA data and associated error values can be found in Supplementary Figures 2 and 3.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Activation Assay, Flow Cytometry, Expressing, Incubation, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 2 | Structural basis for high-affinity binding. (a) Gas6–MYD1 co-complex showing overall architecture and 2:2 stoichiometry. (b) MYD1 Ig1 (orange) and Gas6 LG1 (gray) domains showing the location of the four mutations in MYD1 with respect to the major binding site, which lies at the interface of these two domains. (c) Analysis of the wild-type structure (PDB code 2C5D) reveals steric crowding between the side chains of T457Gas6 and V92Axl. The V92A mutation alleviates this crowding in the MYD1 co-complex and facilitates local reorganization of side chains around V92A, exemplified by R48 and Q94. This in turn creates an elongated groove on MYD1 at the binding interface that allows reorientation of T457 on Gas6. (d) Reorientation of T457 results in capping of the N terminus of helix A. The wild-type (WT, green) and MYD1 (gray) structures are overlaid for comparison. (e) Capping stabilizes helix A, as seen by B-factor analysis (Online Methods).

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 4 | MYD1 Fc inhibits Axl activation and downstream signaling in skov3.ip cells. (a) Wild- type (WT) Axl Fc and MYD1 Fc, but not Axlnb Fc, can inhibit Gas6-mediated Axl activation in vitro. (b) Inhibition of Axl activation leads to reduced levels of phosphorylated Akt and Erk1/2 and an increase in the epithelial marker e-cadherin. For full (uncut) blots, see Supplementary Figure 11.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Activation Assay, In Vitro, Inhibition, Marker

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 5 | Sequestration of Gas6 by MYD1 Fc inhibits metastasis. (a) Amount of free Gas6 in serum of mice 12 h after administration of a single dose of MYD1 Fc. (b) Kinetics of Gas6 sequestration (black) and MYD1 Fc clearance (red) following a 1 mg per kg body weight dose of MYD1 Fc. (c) Using the off-rates of the Gas6-Axl Fc interactions (Fig. 3b), dissociation of Gas6 bound to either wild-type Axl Fc (red) or MYD1 Fc (blue) is plotted over time. The in vivo clearance of the Axl decoy receptors as measured in c is overlaid in black. Two mice were analyzed for each data point in b and c. (d–f) Tumor burden in in vivo models of metastatic human ovarian cancer. The number of visible metastases in animals treated with Axlnb Fc, wild-type Axl Fc or MYD1 Fc was counted in the skov3.ip (d) and OVCAR (f) tumor models. Representative images of mice from each treatment group in the skov3.ip model are shown, and arrows indicate disease (e). In both models, animals were administered 10 mg per kg body weight of the indicated protein twice weekly. (g) Lung metastases in the 4T1 luciferase breast cancer model, as quantified by ex vivo bioluminescent imaging. Mice received intravenous injections of the indicated treatment twice weekly. (h) Representative bioluminescent images of lungs and spleens from each treatment group; scale bar, 1 cm. Error bars represent ± s.d., n = 6–12 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: In Vivo, Luciferase, Ex Vivo, Imaging

Journal: Biomolecules

Article Title: A New Serum Macrophage Checkpoint Biomarker for Innate Immunotherapy: Soluble Signal-Regulatory Protein Alpha (sSIRPα)

doi: 10.3390/biom12070937

Figure Lengend Snippet: Expression and shedding of SIRPα . ( A ) Monocyte derived macrophages (M0) were polarized into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL13 (M2a), IL-10 (M2c) and the expression of SIRPα was investigated using flowcytometry. ( B ) Soluble SIRPα (sSIRPα) was measured in the cell cultures by ELISA. (* p < 0.05, ** p < 0.01). ( C ) Western blotting of serum-samples from four individuals (Lane 2–5: samples, Lane 6: Recombinant extracellular SIRPα).

Article Snippet: A standard curve spanning a range from 0.125-8 μg/L was prepared from a recombinant human SIRPα (R&D Systems, 9378-SA-050).

Techniques: Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Cancer Discovery

Article Title: Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level

doi: 10.1158/2159-8290.cd-21-0316

Figure Lengend Snippet: Figure 3. MRC1+ CCL18+ macrophages in metastatic tumors exhibited terminally differentiated and suppressive states. A, The ranked differential tumor–immune cell cross-talk [liver metastasis (LM) vs. colorectal cancer (CRC)] shows MRC1+ CCL18+ M2-like macrophages ranked the second among all ligand–receptor pairs. B, The liver metastasis upregulated ligand–receptor cross-talk between MRC1+ CCL18+ macrophages and SPP1+ macrophages with cancer cells (liver metastasis vs. colorectal cancer). The y-axis represents the ligand and receptor name, whereas the x-axis represents the tissue. The circle size represents the log-normalized P value, whereas the color darkness represents the log-transformed mean expression of ligand and receptor. C, Multiplex IHC shows the cross-talk between tumor cells and MRC1+ CCL18+ macrophages or SPP1+ macrophages via the ligand–receptor of CD47-SIRPA. D, The volcano plot represents the differentially expressed genes of MRC1+ CCL18+ M2-like macrophages between colorectal cancer and LM. E, The selected gene expression of MRC1+ CCL18+ M2-like macrophages in colorectal cancer and LM. ***, P < 0.005. P values were determined by the Wilcoxon test. F, Pathway enrichment analysis of highly expressed genes of MRC1+ CCL18+ M2-like macrophages in colorectal cancer and liver metastasis, respec- tively. KEGG gene sets were used to perform the pathway enrichment analysis (Methods). Only selected pathways are shown.

Article Snippet: This generated data sets containing 3,826 (ST-P1, liver), 4,658 (ST-P2, liver), 3,695 (ST-P3, liver), 3,721 (ST-P4, liver), 3,313 (ST-P1, colon), 4,174 (ST-P2, colon), 4,007 (ST-P3, colon), and 3,902 (ST-P4, colon) spots. mIHC We performed the fluorescent dyes by using the Osteopontin/SPP1 rabbit anti-human antibody (Abcam; catalog no. ab214050), Ki-67 rabbit anti-human antibody (IBP; catalog no. IR098), CD68 mouse anti-human antibody (Maxim; catalog no. Kit-0026), CCL18 rabbit anti-human antibody (Sino Biological; catalog no. 10502-T16, RRID:AB_2860192), GranzymeK mouse anti-human antibody (ProteinTech; catalog no. 67272-1-Ig, RRID:AB_2882541), GZMB rabbit anti-human antibody (Biolynx; catalog no. BX50024), CD56 rabbit anti-human antibody (IBP; catalog no. IR040), CTLA4 mouse antihuman antibody (Origene; catalog no. TA810299), CD45 mouse antihuman antibody (BioLegend; catalog no. 304002, RRID:AB_2661811), CD15 mouse anti-human antibody (Maxim; catalog no. MAB-0779), DAPI (BioLegend; catalog no. 422801), MIF polyclonal rabbit antihuman antibody (Abcam, ab65869), GSTO1 rabbit anti-human antibody (Abcam, ab129106), IL4I1 rabbit anti-human antibody (Abcam, ab222102), SIRPA mouse anti-human antibody (OriGene, TA807751), CD47 rabbit anti-human antibody (Abcam, ab226837), and cytokeratin Pan (Pan Ck) mouse anti-human antibody (LBP Medicine, IHCM067).

Techniques: Transformation Assay, Expressing, Multiplex Assay, Gene Expression

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: Exposure to allergen upregulates IL-27 in murine MACs during CHS. (A) Quantitative analysis of CD45 + hematopoietic cell frequency (in living cells) in DNFB-treated and vehicle-treated ear skin of the CHS mice (2 and 7 days post-DNFB elicitation). The data are represented as mean ± standard error of the mean (SEM) from at least 4 mice per group, *p < 0.05 (unpaired Student’s t test). (B) Gating strategies for skin myeloid cell population identification in the CHS mouse model. Single-cell suspensions of mouse ear skin treated were prepared. After excluding dead cells, as well as Lin + ; including T cells, NK cells, B cells, and granulocytes, the remaining CD45 + cells were analyzed for expression of CD24 and CD11b. CD11b + CD24 -/lo cells were further analyzed for Ly-6C, CD64, and CCR2 expression. The CD11b + non-DCs fraction was separated into monocyte and macrophages (MACs) populations, respectively. (C) Pie charts summarizing immune cell distribution (gated on CD45 + Lin - cells) from mouse ear skin at 2 and 7 days post-DNFB elicitation. The data represent the mean of at least 4 mice per treatment group. (D) Histogram from representative flow cytometry analysis for IL-27p28 of vehicle-treated and DNFB-treated ears gated on CD45 + cells. Data shown are representative of at least 4 mice per group. (E) Data presented shows median fluorescence intensity (MFI) of IL-27p28 in the CD45 + population from ll-27p28 EGFP mice at 7 days post DNFB-elicitation versus vehicle controls from at least 4 mice per group and are depicted as mean ± SEM, *p < 0.05 (unpaired Student’s t test). (F) Quantitative analysis of CD172a + IL-27p28 EGFP+ cell frequency of CD45 + Lin - CD11b + cells in DNFB-treated and vehicle-treated ear skin. At least 4 mice per group and summarized as mean ± SEM, *p < 0.05 (unpaired Student’s t test). (G) Representative flow cytometric overlay dot plots of gated monocytes, MACs, and CD11b + DCs at 7 days post-DNFB elicitation on mouse ear skin demonstrating CD172a and IL-27p28 expression from at least 4 mice per group.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: CD3, CD47, and CD172a expression in human ACD clusters. (A–C) Immunofluorescence staining of CD3 (green), CD47 (red), CD172a (purple), CD14 [green, a serial slide section with the staining of CD172a (purple)], and Hoechst (blue) in human donor-matched patch-test negative control and patch-test (+) ACD skin. White dashed lines mark the epidermal-dermal junction. Data are representative of 3 patient samples per tested condition. (A) Scale bars are 200 µm (left) and 100 µm (right). (B, C) Scale bars are 20 µm.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control

Journal: Nature Communications

Article Title: Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

doi: 10.1038/s41467-024-54129-w

Figure Lengend Snippet: a Mechanism of action of conventional aEGFRvIII CAR T cell monotherapy in GBM. b Proposed aEGFRvIII-SGRP CAR T cell combination therapy whereby SGRP-mediated CD47 blockade induces phagocytic modulation of GAMs in the context of EGFRvIII-heterogenous GBM and its immunosuppressive iTME. c Outline of the SGRP engineering strategy, including specific AA substitutions to the endogenous human SIRPγ-V1 sequence and addition of an N-terminal IL-2 signal sequence (IL2sig) leading to constitutive SGRP secretion. d Polycistronic lentiviral constructs encoding mCherry (mC)-labeled aCD19 CAR or aEGFRvIII CAR under the control of EF1A promoter ± SGRP secretion. e Workflow of CAR T cell production applied throughout the study. a – e Created in BioRender. Hutter, G. (2022) BioRender.com/u48r093. Representative plots of CAR:target protein binding by aCD19 CAR T cells ( f ) or aEGFRvIII CAR T cells ( g ) to CAR-bound biotinylated (bt)-CD19 (top plots) or bt-EGFRvIII (bottom plots); n = 2 healthy donors (HDs) assessed per CAR. h TATA-box binding protein (TBP)-normalized expression of mCherry and SGRP detected by real-time quantitative PCR (RT-qPCR) in aEGFRvIII CAR or aEGFRvIII-SGRP CAR T cells, showing mCherry expression in CARs transduced with either construct and SGRP expression specifically in aEGFRvIII-SGRP CARs; n = 4 HDs. Data are presented as scatter plots with mean values ± SD. Statistical differences were assessed by two-sided unpaired t tests with Welch’s correction. i Differentially secreted proteins in aEGFRvIII-SGRP CAR- vs aEGFRvIII CAR-conditioned media, highlighting the presence of SGRP exclusively in aEGFRvIII-SGRP CAR; n = 2 HDs. Mean SGRP expression: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-\log 10{{{\rm{qValue}}}}/\log 2{{{\rm{foldchange}}}}=8.60/6.65$$\end{document} − log 10 qValue / log 2 foldchange = 8.60 / 6.65 . Source data are provided as a Source Data file. Source data for ( i ) are provided as Supplementary Data .

Article Snippet: Experiments involving ELISA assays were performed using a CD47:SIRP alpha Biotinylated Inhibitor Screening ELISA Assay Pair (#EP-102, ACROBiosystems, USA) or Human SIRP alpha DuoSet ELISA (#DY4546-05, R&D Systems, USA).

Techniques: Sequencing, Construct, Labeling, Control, Protein Binding, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction

Journal: Nature Communications

Article Title: Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

doi: 10.1038/s41467-024-54129-w

Figure Lengend Snippet: a Assessment of CAR T cell on-target killing capacity by co-culture time-lapse of nEGFP + U251vIII with mCherry + target-specific (aEGFRvIII CAR ± SGRP) or nonspecific (aCD19 CAR ± SGRP) at a 1:1 E:T ratio for 72 h. b Assessment of CAR T cell off-target killing by co-culture time-lapse of nEGFP + U251 with mCherry + CAR T cells at a 1:1 E:T ratio for 72 h. c Dose-dependent CAR T cell killing capacity in a co-culture with U251vIII at defined time points. Dashed lines represent the mean confluence in control wells with only U251vIII cells. a – c Curves and dots represent the mean of duplicate measurements. d Representative histograms of CAR T cell degranulation in 24 h co-cultures with U251vIII, BS153, U251, or U87 (gated on live mCherry + singlets); Conditions were performed in duplicates with n = 2 HDs. e IFNγ release detected by ELISA in supernatants of CAR T cells co-cultured with EGFRvIII + or EGFRvIII - GBM cell lines. Conditions were performed in triplicates with n = 2 HDs. f Schematic illustration of the experimental setup of an SGRP/aCD47 blocking assay on CD47 + BS153 cells. Created in BioRender. Hutter, G. (2024) BioRender.com/i60q967. g Scatter plots of the MFI of individual wells. Data are presented as mean values ± SD. Conditions were performed in triplicates. Statistical differences were assessed by two-sided unpaired t tests with Welch’s correction. Source data are provided as a Source Data file.

Article Snippet: Experiments involving ELISA assays were performed using a CD47:SIRP alpha Biotinylated Inhibitor Screening ELISA Assay Pair (#EP-102, ACROBiosystems, USA) or Human SIRP alpha DuoSet ELISA (#DY4546-05, R&D Systems, USA).

Techniques: Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Blocking Assay