Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4+ T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. (B) 24 h co-cultures of SEB Ag-presenting DC1 and CD4+ T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). (C) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. (D) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel). Individual segments begin at an origin or branch point (orange spheres) and end at the next branch or terminal point (green spheres). Spatial ‘reach’ was defined as the shortest distance from a filament origin to the distal terminal point of that filament (right panel; white lines connecting 2 points). (E) Total cell surface area comparisons of resting and CD40L-activated DC1 and DC2. (F) Comparison of percentage ‘TNT positive’ resting DC1 and DC2 (media only), defined as those expressing ≥5 individual TNTs per cell that were each >5.0 μm in length. (G) Comparison of percentage ‘reticulation positive’ CD40L-activated DC1 and DC2, delineated as those displaying ≥5 filaments per cell that were each >10.0 μm in sum segment length. (H) ‘Maximum reach’ of filaments, defined as the shortest distance (μm) from the origin to the farthest terminal point of a filament. (E-H) Data were generated from randomly chosen image fields (20–30 per donor) and represented as mean ± SD of 3 healthy donors independently tested. P-values <0.0001, <0.001, <0.01 and <0.05 are represented by ****, ***, **, and *, respectively. See also Supplemental Fig. 1; Video 1; Video 2.

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Cell Culture, Control, Blocking Assay, Membrane, Labeling, Comparison, Expressing, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A–C) 3D reconstruction images (600×) of differentially matured DC stimulated for 20 h with rhCD40L prior to labeling the cell surface with MHC class I mAb (green) and labeling nuclei (blue), followed by live-cell confocal imaging. Data are representative of 6 independent experiments conducted using DC from 3 healthy donors. (A) Membrane morphologies of mature CD40L-activated DC0, DC1, and DC2 propagated using LPS alone, LPS + IFN-γ, or LPS + PGE2, respectively. (B) Mature CD40L-treated DC0, DC1, and DC2 generated by the respective use of R848 alone, R848 + IFN-γ, or R848 + PGE2. (C) Cell morphologies of DC1 treated with media alone or CD40L. DC1 were generated using the αDC1 cytokine-based cocktail (first two panels on left), or induced by 48 h co-culture of iDC with either 2-signal activated NKh cells (middle) or SEB-activated CD8+ T cells (right).

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Labeling, Imaging, Membrane, Generated, Co-Culture Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) Graphical display of the percentage of reticulation positive TNF-α-matured DC0 after 20 h exposure to rhCD40L alone, or in combination with IL-4, IL-10, or IFN-γ. Also shown are representative confocal images (600×) of DC0 stimulated with rhCD40L alone or in combination with IFN-γ and then surface labeled with MHC class I-specific mAb (green) and nuclear labeled (blue). (B) Graphical depiction of percentage reticulation positive αDC1 after 20 h exposure to rhCD40L alone, or combined with IL-4, IL-10, or IFN-γ. Additionally shown are representative confocal images (600×) of DC1 stimulated with rhCD40L alone or in combination with IL-4. (A-B) Quantitative data, displayed as percentage positive ± SE, are representative of 3 healthy donors independently tested twice per donor. See also Supplemental Fig. 2.

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) Sequential still frames of DC1 actively reticulating from 4 to 7.6 h post-addition of rhCD40L, captured by live-cell, time-lapse DIC imaging (600×). See also Video 3. (B) Sequential frames of high resolution, time-lapse DIC imaging (600×) of live 8 h rhCD40L-stimulated DC1 showing endogenous cell structures resembling vesicles (arrows) trafficking between neighboring cells through CD40L-induced TNTs. See also Video 4. (C) Confocal reconstruction images (1000×) revealing early endosome- (green, arrows) and F-actin-containing TNTs (red) and nuclei (blue) in fixed CD40L-activated DC1. Data are representative of 3 donors independently tested.

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A–C) Cy5-labeled (blue), YG latex bead (40 nm)-containing ‘donor’ DC1 or DC2 were co-cultured with the respective non-bead-containing, Cy3-labeled (red) ‘recipient’ DC type for 20 h in the presence or absence of rhCD40L. Data are representative of 2 donors independently tested. (A) Montage of time-lapse, confocal imaging (600×) showing beads (green; arrows) moving rapidly through donor DC1 TNTs (blue) over a time span of 28 min. See also Video 5. (B) Montage tracing the pathway of beads (green; arrows) traveling from the cell body of donor DC1 (blue), through donor cell TNTs and into the connected recipient DC (red), where they finally collect in the recipient cell body (yellow, lower left arrows). (C) Flow cytometric analysis and quantification of intercellular exogenous bead transfer from donor to recipient DC types after treatment with media only or CD40L. (D) IFNγ-ELISPOT assays measuring Ag-specific recall responses of cultured T cells following their in vitro sensitization with FACS sorted, YG bead/Ag positive and negative recipient DC1. Data are represented as mean ± SE of 2 independent experiments, and * represents the p-value <0.05. See also Supplemental Fig. 3.

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Labeling, Cell Culture, Imaging, Enzyme-linked Immunospot, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) DC1 monolayers that had been stimulated with rhCD40L for 10 h were exposed to EGFP-expressing Escherichia coli for 2 h. Subsequent time-lapse confocal resonant scanning microscopy shows individual bacterium (green) trafficking bi-directionally along CD40L-induced TNTs (arrows) between mature DC1. See also Video 6. (B) Donor DC1 (blue) pulsed with EGFP-expressing HIV-1-like particles (green) were co-cultured with recipient DC1 (red) in the presence of CD40L for 10 h prior to live-cell imaging. HIV-1-like particles localizing to a donor cell nanotube (blue; arrows) that has formed a connection with a recipient cell body. (C) At 20 h post-CD40L stimulation, HIV-1-like particles can be detected at the interface between blue donor cell TNTs and red recipient cells (arrows), prior to their subsequent transfer to recipient cell bodies. (A-C) Imaging data are representative of 3 donors independently tested.

Article Snippet: Importantly, the formation of these membrane bridges required the presence of Ag as well as CD4 + T cells, and did not occur in co-cultures containing CD8 + T cells (data not shown). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 CD4 + Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1 ( A ) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4 + T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. ( B ) 24 h co-cultures of SEB Ag-presenting DC1 and CD4 + T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). ( C ) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. ( D ) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel).

Techniques: Expressing, Microscopy, Cell Culture, Live Cell Imaging, Imaging