Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigens

doi: 10.1073/pnas.2016857118

Figure Lengend Snippet: H57-scFV-induced T cell activation depends on ligand spacing. (A) Pseudo-colored images of T cells recorded in a typical calcium experiment for mSA-H57 and M-H57 at a ligand density of ∼4 µm−2 (Left). Corresponding pseudo-colored calcium flux traces of >100 cells per region (Right). Cell traces (each horizontal line is one cell) are ranked by their integrated calcium flux in descending order. Calcium was imaged every 1 s. (Scale bar, 4 µm.) (B) Dose–response curves for T cell activation at different ligand surface densities. Each data point corresponds to the percentage of activated cells determined in an individual experiment at a specific ligand density. Data were normalized to a positive control (=100%) containing His-tagged ICAM-1 and B7-1 at 100 µm−2 and His-tagged pMHC (His-pMHC) at 150 µm−2. Dose–response curves were fitted with Eq. 6 (see Materials and Methods) to extract activation thresholds (C). For each dose–response curve, data are from at least three independent experiments and three different mice. For details, refer to SI Appendix, Table S3. Error bars represent the 95% CI. ***P < 0.001; n.s., not significant. A matrix containing the results of significance tests for all combinations of ligands is shown in Dataset S1. (D) ZAP-70–recruitment to the T cell plasma membrane at a ligand density of ∼50 µm−2. T cells were fixed 10 min after cell seeding and immunostained for ZAP-70. The signal was analyzed and normalized using a positive control (as in B) as a reference. Cells on bilayers containing only ICAM-1 and B7-1 at 100 µm−2 are shown as a negative control. Data are shown as medians ± CI, with each data point representing a single cell. n > 50 cells from at least three independent experiments and three different mice. ***P < 0.001; **P < 0.05; n.s., not significant. A matrix containing the results of significance tests for all combinations of ligands is shown in Dataset S2. (E) Representative TIRF images for the different constructs featuring AF555-labeled ligands at densities of ∼4 µm−2 (indicated by a vertical dashed gray line in B). The cell outline is indicated by a dashed white contour line. Images were recorded 10 min after seeding. Dy548-labeled DNA origami platforms without ligands at ∼4 µm−2 are shown for comparison. (Scale bar, 2 µm.)

Article Snippet: Finally, His 10 -tag ICAM-1 (50440-M08H, Sino Biological) (270 ng ⋅ mL −1 ) and His 10 -tag B7-1 (50446-M08H, Sino Biological) (130 ng ⋅ mL −1 ) were incubated for 75 min at 24 °C and then rinsed off with PBS.

Techniques: Activation Assay, Positive Control, Negative Control, Construct, Labeling

Journal: bioRxiv

Article Title: Intraperitoneal programming of tailored CAR macrophages via mRNA-LNP to boost cancer immunotherapy

doi: 10.1101/2024.07.30.605730

Figure Lengend Snippet: In situ programming of CAR-M leads to M1 polarization with enhanced PD-L1 and MHC I expression. a, Schematic illustration of scRNA-seq analysis of the CT26-luc syngeneic mouse model. b, Proportion of tumor-infiltrating immune cells labeled via scRNA-seq. c, UMAP plot of scRNA-seq profiles from macrophages within the tumor. d, Counts of clustered macrophages in groups. UMAP plot of relative expression for the indicated gene (e, TREM2; f, NOS2; g, CD80; h, CD274). i, Schematic representation of CAR signal transduction leading to upregulated PD-L1 and MHC I expression. j, Flow cytometry analysis of macrophage PD-L1 and H-2Kd expression in groups. Mean fluorescence intensity (MFI) was calculated from flow cytometry profiles of macrophage PD-L1 (k) and H-2Kd (l). Statistical significance was calculated using one-way ANOVA. For all panels, ns = no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. m, In vivo bioluminescent imaging of the CT26-luc syngeneic mouse model treated with UTD, PD-L1 siRNA, CAR-M, and combination therapy. n, Heatmap of featured genes of clustered tumor-infiltrating T cells. o, Counts of clustered T cells in groups. p, Schema of the mechanism of potent synergy between tailored CAR-M and ICB.

Article Snippet: Primary antibodies used in the study are listed as follows: F4/80 (123117, BioLegend), CD80 (104715, BioLegend), CD80 (50446-R014-A-25, Sino Biological), CD86 (105005, BioLegend), CD86 (50068-RP02-50, Sino Biological), CD206 (FAB2535P, R&D), CD206 (#24595, CST), CD3 (100228, BD), CD4 (550954, BD), CD8 (K0227-A64, MBL), IFNr (505807, BioLegend), and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, abcam).

Techniques: In Situ, Expressing, Labeling, Transduction, Flow Cytometry, Fluorescence, In Vivo, Imaging

Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A) Representative 3D-SIM images of OT1 T cells fixed 7 min after surface attachment and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80, G4:H-2K b plus CD80, CD80 only, or streptavidin (SA) only. The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (B) Examples for FibrilTool analysis. 7–8 similar-sized trapezoidal ROIs covering the pSMAC portion of the IS were drawn to measure the anisotropy of actin arcs. (C) Average anisotropies of actin arcs in the pSMAC region of OT1 T cells activated on surfaces coated with OVA, G4, or CD80-only. (D) Total anisotropies of actin arcs from all ROIs measured in OT1 T cells activated on surfaces coated with OVA, G4, and CD80-only. (E) Histogram of total anisotropies from all ROIs measured on OVA- and G4-coated surfaces (p < 0.00001). Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. ***, **** indicate p < 0.001, 0.0001.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining, Labeling

Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80 (A) or G4:H-2K b plus CD80 (B). The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (C) Frequency of arc morphologies in OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Arcs were scored as either Organized (i.e. present and concentric), Disorganized (i.e. present but either pointing inwards or entangled), or No arcs (i.e. not present). (D) Average anisotropies of actin arcs in the pSMAC region of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. (E) Histogram of total anisotropies from all ROIs measured on OVA-coated surfaces with or without pnBB treatment (p < 0.0001). (F) Same as (E) but using G4-coated surfaces (p = 0.44). Mean ± SEM. **** indicates p < 0.0001.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining, Labeling

Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-pCasL antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80 (A) or G4:H-2K b plus CD80 (B). Scale bar, 5 μm. (C) Mean intensities of pCasL at the IS from 3D-SIM images of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. * and ** indicate p < 0.05 and < 0.01, respectively.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining