Journal: Nature Communications

Article Title: T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells

doi: 10.1038/s41467-018-06090-8

Figure Lengend Snippet: T cells generate microvillus-originated particles upon TCR stimulation. a T cell microvillus polarization toward antigen-bearing B cells at the early stage of IS. SEM of resting single (left) or two adjacent (right) Jurkat T cells on PLL (i) and co-incubated with SEE-loaded Raji B cells (5, 30 min) (ii). Arrows indicate microvilli (red) and membrane ruffles (blue). b , c SEM of OTII naive CD4 + T cells on PLL with the anti-CD3 antibody ( b ) or OVA peptide/I-A b and ICAM-1 ( c ) at various time points. Early (early stage, 1 min), mature (1–5 min), late (10–20 min), and terminal stages ( > 20 min). d Jurkat T cells were placed on anti-CD3-coated coverglass for 30 min and observed by TEM. The areas of TMPs and mitochondria were quantitated using ImageJ software. * P < 0.01 vs. mitochondria

Article Snippet: Antibodies against TCRα (sc-9100), TCRβ (sc-73121), CD3ζ (sc-1239), CD3ε (sc-137095), CXCR4 (sc-53534), CD43 (sc-7054), and MHC class I (sc-59199) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Membrane, Software

Journal: Nature Communications

Article Title: T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells

doi: 10.1038/s41467-018-06090-8

Figure Lengend Snippet: TCR-enriched microvillus particles are released to a greater extent during T cell kinapse mode. a Co-localization of V5G with TCR microclusters. V5G + OTII CD4 + T cells were stained with anti-TCRβ (H57Fab-Alexa594) and examined on a planar bilayer presenting OVA peptide/I-A b and ICAM-1 (see also Supplementary Movie ). Relative time scales starting from the time of cell spreading are labeled on the images. Representative trajectory of individual TCR microclusters are shown in the right panel. b V5G + OTII CD4 + T cells were pretreated for 30 min with (Lat A; 237 nM), JAS (100 nM), and CK636 (100 μM), followed by SEM (left) and TIRFM imaging to visualize V5G + microvillus movement on the lipid bilayer (middle). Boxed regions are shown enlarged at the bottom of the panels. Representative trajectories of individual V5G clusters (bottom). Statistical analyses of the frequency of cSMAC formation and the number of V5G clusters in OTII CD4 + T cells (right). * P < 0.05 vs. none. Data represent the mean of three experiments ± SEM. c , d Release of TCR + TMPs during synapse and kinapse. V5G + OTII CD4 + T cells were stained with anti-TCRβ (H57Fab-Alexa594) and observed on a lipid bilayer presenting OVA peptide/I-A b and ICAM-1 by TIRFM (see also Supplementary Movie ). The numbers of TCRβ + TMPs were quantitated using ImageJ software ( c ). * P < 0.01 vs. no antigen (No-Ag). Data represent the mean of three experiments ± SEM. e L- and S-TMP release during synapse and kinapse. Cells from ( d ; synapse–kinapse) were subjected to SEM analysis. P, pSMAC; C, cSMAC. Particle diameters were analysed using 500 particles in the C and P of a single cell, and > 30 cells were examined. Data represent the mean of three experiments ± SEM. * P < 0.001 vs. C. f TMP generation from OTII CD4 + T cells was examined in each condition. Cells were subjected to SEM, and the number of TMPs per cell was analysed using ImageJ software. Data represent the mean of three experiments ± SEM. * P < 0.001 vs. PLL

Article Snippet: Antibodies against TCRα (sc-9100), TCRβ (sc-73121), CD3ζ (sc-1239), CD3ε (sc-137095), CXCR4 (sc-53534), CD43 (sc-7054), and MHC class I (sc-59199) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Staining, Labeling, Imaging, Software

Journal: Nature Communications

Article Title: T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells

doi: 10.1038/s41467-018-06090-8

Figure Lengend Snippet: L-TMPs are further fragmented by membrane-budding complexes. a Localization of Arrdc1 and TSG101 in the microvillus tips of HEK293T cells. Arrdc1_GFP was represented with pseudo-color coding according to fluorescence intensity. Cropped images from selected microvilli (yellow arrows) are displayed from i to v (left). Recruitment of TSG101 to the membrane in Arrdc1 -overexpressing cells (right). b Co-clustering of Arrdc1_GFP and TCRβ into the cSMAC. Arrdc1_GFP + OTII CD4 + T cells were stained with anti-TCRβ (H57Fab-Alexa594) and examined as described in Fig. . Images were selected at 300-s post-recording, and the representative trajectories are shown in the bottom panel. c Release of TCR + /Arrdc1_GFP + TMPs during the synapse–kinapse transition stage. Arrdc1_GFP + OTII CD4 + T cells were stained with anti-TCRβ and observed as described in Fig. . d Purified TMPs were ultracentrifuged with a sucrose gradient and blotted with anti-Arrdc1, anti-CD3ζ, and anti-β-actin (lanes 1–6, sucrose fraction; P, pellet). e Knockdown of Vps4 and Arrdc1 by siRNA and confirmation of siRNA efficiency by western blot analysis. Surface analysis of CD4 + T cells by SEM after knockdown of the indicated proteins. f , g Representative SEM analysis of Vps4 -deficient OTII CD4 + T cells on the lipid bilayer ( f ). Particle diameters and numbers of TMPs per cell were analysed by ImageJ software ( g ). Data represent the mean of three experiments ± SEM. * P < 0.05 vs. scrambled (Sc., left) or scrambled (20–40 nm, right) siRNA. ** P < 0.001 vs. scrambled siRNA (40–100 nm). h TCR + TMP release on an OVA peptide/I-A b lipid bilayer with or without ICAM-1. The number of TCR + TMPs per cell was analysed by ImageJ software. Data represent the mean of three experiments ± SEM. * P < 0.001 vs. pMHC/ICAM-1. i TCR + TMP release on plates coated with various antibodies. Data represent the mean of three experiments ± SEM. * P < 0.001 vs. PLL

Article Snippet: Antibodies against TCRα (sc-9100), TCRβ (sc-73121), CD3ζ (sc-1239), CD3ε (sc-137095), CXCR4 (sc-53534), CD43 (sc-7054), and MHC class I (sc-59199) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Membrane, Fluorescence, Staining, Purification, Knockdown, Western Blot, Software

Journal: Nature Communications

Article Title: T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells

doi: 10.1038/s41467-018-06090-8

Figure Lengend Snippet: Physical interaction between TMPs and the DC surface is critical for TMP-mediated DC activation. a Schematic diagram of the transwell system (left). After co-incubation for 2 h, DCs were acid washed and evaluated for TCRβ and V5G transfer (upper panel), followed continued incubation for 24 h. The expression of MHC class II, CD80, CD86, and CD40 on DCs (CD11c + ) was analysed by flow cytometry and expressed as histograms and mean fluorescence intensity (MFI). Data represent the mean of three experiments ± SEM. * P < 0.01 vs. c . b 4-TMPs from anti-CD3-coated surfaces were treated with DCs as depicted in the schematic diagram (left) and incubated as described in a . TCRβ, CD86, and CD40 presentation on DCs (CD11c + ) was analysed as described in a . Data represent the mean of three experiments ± SEM. * P < 0.01 vs. c . c Transfer of TCRs to DCs in vivo. OTII TCR transgenic mice were administered OVA257-264 and OVA323-339 (100 μg; i.p.) in PBS, respectively. After 48 h, draining lymph nodes were taken and dissociated, and cells were treated by acid buffer and stained for the indicated surface markers. Boxed areas represent TCRβ expression on DCs. Data are represented by one of three independent experiments with similar results. * P < 0.01 vs. none or OVA257-264

Article Snippet: Antibodies against TCRα (sc-9100), TCRβ (sc-73121), CD3ζ (sc-1239), CD3ε (sc-137095), CXCR4 (sc-53534), CD43 (sc-7054), and MHC class I (sc-59199) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Fluorescence, In Vivo, Transgenic Assay, Staining