anti icam 1 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) Schematic diagram of a T cell-hydrogel contact on glass (left) with DAAM-particle functionalization strategy (right). (B) Left, representative 3-D projection (left) of a Lifeact-eGFP expressing OT-1 CTL interacting with a H-2K b -OVA/ICAM-1 coated DAAM particle (magenta). Right, super-localized triangulations of the particle are shown, with triangles shaded by mean curvature in the image at the far right. Positive (convex) curvatures are shaded blue-green while negative (concave) curvatures are shaded gold. (C) Representative world map projection of the triangulated particle in B, with mean curvature shown to the left and CTL Lifeact-eGFP intensity within 1 µm of the particle surface shown to the right. Regions of interest (ROIs, dotted lines) were defined by the boundary of substantial Lifeact-eGFP intensity. (D) Left, cross-section at the center of a representative CTL-bound particle with contact area labeled in blue. Right, corresponding radial profile about the perimeter of the particle. (E) Integrated compressed volume (left) and estimated compression force (right) of CTL-DAAM particle contacts compared to non-contact areas on particles (n = 61 synapses, n = 56 unindented spheres). **** denotes P ≤ 0.0001, calculated by unpaired Welch’s t-test. Error bars indicate standard deviation (SD). (F) Particle distortion and force estimates over time from a representative time-lapse in which contact is initiated at t ≈ 5 min (see Movie S1). (G) Temporal autocorrelations of mean curvature profiles within the synapse ROI, determined from 19 time-lapse videos. Blue traces were constructed from time-lapses with 3-minute/frame intervals, while red traces were constructed from 15 second/frame videos.

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: Expressing, Labeling, Standard Deviation, Construct

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) Synapses were partitioned into radial bins starting from the center of the contact and proceeding to the periphery. A representative synapse is shown, with positive curvature in blue-green and negative curvature in brown. (B) Curvature plot derived from the synapse in A. Vertical strips contain the probability distributions of curvatures within each radial bin, proceeding from the center (left) to the periphery (right). (C) Mean radial curvature plots derived from CTL synapses (n = 61, left) and non-synapse control surfaces (n = 56, right). Representative topographies are shown in the top left of each plot. (D) Difference plot determined by subtracting the non-synapse curvature distribution from the CTL curvature distribution. Curvature domains are colored red and blue if they are over-represented in CTL synapses and non-synapse controls, respectively. Dotted line indicates 0 curvature (flat). (E-G) Time-lapse montages of a representative Lamp1-eGFP expressing OT-1 CTL interacting with an α-CD3ε/ICAM-1 coated DAAM particle. (E) Side view of the degranulation event. (F) En face view of Lamp1-eGFP within 0.5 µm of the particle surface. In E and F, yellow arrowheads denote a lytic granule that docks at the cell-target interface and disappears, indicating degranulation (t = 3 min). (G) Cropped world map projection of the underlying particle surface, shaded by mean curvature. The degranulation zone is indicated by red arrowheads. (H) Mean curvature (black line ± SD in gray) plotted as a function of normalized distance from the center of the contact. Mean curvatures within a 1 µm diameter zone around each degranulation event were calculated and plotted against radial distance as red X’s (n = 15 cells with n = 17 degranulation events).

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) Schematic diagram highlighting the cytoskeletal proteins targeted by CRISPR/Cas9 in this data set. WASp and WAVE2 drive protrusive actin polymerization, while Talin couples integrins to the F-actin cytoskeleton. (B-D) OT-1 CTLs expressing Cas9 were transduced with retroviruses expressing the indicated sgRNAs or non-targeting control gRNA (sgNT), and then imaged together with DAAM particles coated with pMHC and ICAM-1. (B) Cropped views of representative synapses from each experimental group. (C) Deformation volume of CTL-DAAM particle contacts (n = 252 cells). ** denotes P ≤ 0.01, calculated by multiple t-test with Tukey’s correction. Error bars denote SD. (D) Difference plots of radial curvature distribution, obtained by subtracting the mean curvature distributions of the indicated mutant CTLs from the mean curvature distribution of sgNT control CTLs. Curvature domains are colored red and blue if they are over-represented in sgNT and mutant CTLs, respectively. Dotted line indicates 0 curvature (flat).

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: CRISPR, Expressing, Transduction, Mutagenesis

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) OT-1 CTLs and 5C.C7 T H Cs were cultured in parallel and imaged together with DAAM particles coated with anti-CD3ε antibody and ICAM-1. (B-E) Quantification of particle compression (B), protrusion number (C), peak indentation concavity (D), and peak relief convexity (E) within synapses formed by the indicated T cells. (F) Difference plot of radial curvature distribution, colored red and blue for over-representation of CTLs and T H Cs, respectively. Dotted line indicates 0 curvature (flat). (G) CTL and T H C topographies were represented as Z-pattern spectra and visualized by PCA. Data points were separated into terciles (indicated by the blue lines) for downstream analysis. A) Zernike polynomial loading of PC1. Modes with particularly low contributions are highlighted. (I) Cropped views of representative synapses from each tercile in G. (J) Distribution of CTL and T H C synapses across the terciles of PC space. (K) Spectral complexity comparison of CTLs and T H Cs. **, ***, and **** denote P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001, respectively, calculated by unpaired Welch’s t-test. All error bars indicate SD.

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) Protocol for i n vivo differentiation of CD8 + T mem , T exh , and T eff cells expressing the P14 TCR. All three cell types, together with naïve P14 T cells, were isolated concurrently and imaged together with DAAM particles coated with anti-CD3ε and ICAM-1. (B) Deformation volume generated by each T cell subset (n = 944). (C) Synapse topographies were transformed into Z-pattern spectra and then visualized by PCA. Data points were separated into quartiles (divided by the blue lines) for downstream analysis. (D) Cropped views of representative synapses from each quartile in D. (E) Distribution of each P14 subset across the quartiles of PC space. (F) Spectral complexity metrics for all P14 subsets. In B and F, ** and **** denote P ≤ 0.01 and P ≤ 0.0001, respectively, calculated by multiple t-testing with Tukey’s correction. Error bars indicate SD.

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: Expressing, Isolation, Generated, Transformation Assay

Journal: bioRxiv

Article Title: Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

doi: 10.1101/2023.04.16.537078

Figure Lengend Snippet: (A) OT-1 CTLs and C57BL/6 BMDMs were cultured in parallel and imaged together with anti-CD3ε/ICAM-1 coated and IgG-coated DAAM particles, respectively. (B) Cropped views of representative BMDM and CTL contacts, colored for curvature. (C) Cross-sectional view of representative BMDM and CTL contacts, with contact area colored red and blue, respectively. Gray dotted line indicates the footprint of an unperturbed sphere. (D) Deformation volume of BMDM (n = 109) and CTL contacts (n = 96). (E) Difference plot of radial curvature distribution, colored red and blue for over-representation of BMDMs and CTLs, respectively. Dotted line indicates 0 curvature (flat). (F-H) Quantification of protrusion number (F), peak indentation concavity (G) and peak relief convexity (H) within BMDM and CTL contacts. (I) BMDM and CTL topographies were transformed into Z-pattern spectra and then visualized by PCA. Representative topographies from each PCA region have been overlaid onto the plot. ns, ***, and **** denote P > 0.05, P < 0.001, and P < 0.0001, calculated by unpaired Welch’s t-test. All error bars indicate SD.

Article Snippet: For T cell assays, 2 × 10 5 ICAM-1 + beads were adhered overnight onto 170-μm imaging coverslips (Ibidi μ-Slide 8 Well) coated with biotin-poly-L-lysine, blocked with 0.5 % (m/v) bovine serum albumin in PBS, coated with 10 µg/mL streptavidin in blocking buffer, and then coated with 1 μg/mL biotinylated rat anti-ICAM-1 antibody (clone YN1/1.7.4, eBioscience, TFS 13-0541-85).

Techniques: Cell Culture, Transformation Assay