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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: J Immunol. 2014 Dec 29;194(3):1047–1056. doi: 10.4049/jimmunol.1401832

Figure 1. CD4+ Th cell-associated CD40L induces TNT-like protrusions that greatly increase the surface area and spatial reach of individual DC1.

Figure 1

(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.