Visualization of T-cell–dendritic cell (T-DC) virological synapses by superresolution fluorescence microscopy. (A and B) STED microscopy of conjugates of dendritic cells pulsed with ATTO-647N–labeled HIV (red) and autologous CD4⁺ T cells labeled with anti-CD3 antibody (A) or with fluorescent phalloidin (B), which is a marker for actin distribution (green). (C and D) Expanded view of the boxed region in B showing comparison of images recorded using conventional confocal microscopy (C) or STED microscopy (D). (E) Fluorescence intensity profiles across synapse (arrows in C and D) show that the spatial resolution of STED imaging (red line, peak marked with asterisk) is better than that of confocal imaging (orange line for HIV, green line for phalloidin) and is adequate to detect single virions (marked by the downward arrow in E and by the yellow circle in D) not resolved with conventional confocal imaging of the same field. (F) STED image of conjugates labeled as in A but in the presence of cytochalasin D, added during synapse formation, illustrating that viruses are no longer clustered or localized at the synapse under these conditions (HIV, red; anti-CD3 antibody, green). Simultaneous imaging of ATTO-647N–labeled HIV-1 (G; red) and lysosomal marker LAMP-1 (H, green) shows that that they display nonoverlapping distributions (I). Pearson's correlation coefficient between fluorescent signals derived from HIV and LAMP-1 dropped to 0.29 with STED imaging as compared with 0.34 with confocal imaging of the same region. (Scale bars: A, B, and F, 3 μm; C, D, G, and H, 1 μm.)

IA-SEM and electron tomography of contact regions between dendritic cells and T cells illustrating HIV-1 distribution within the virological synapse. (A) Interdigitation of membrane protrusions from the T cell (shown in yellow) into the dendritic cell (shown in pink) surface and connection of virion-rich compartments in the interior of the dendritic cell to the synapse via HIV-containing channels (red circle; Movie S3). (B–D) Tomographic slices obtained from ~200-nm thick sections of cocultures of HIV-1–pulsed dendritic cells (DC) mixed with CD4⁺ T cells (T) illustrating different structural aspects of the cell-cell contacts that define the synapse. (E–G) Segmentation of regions boxed in B–D to indicate membrane contact and virus (red) location. (Scale bar: A, 1 μm; B–D, 0.4 μm.)

Model for virus internalization, formation of virion channels, and transfer of virions from antigen-presenting cells to T cells at the virological synapse. (A and B) Sheet-like processes on the surfaces of dendritic cells fold back onto the membrane surface, entrapping viruses in the vicinity into compartments that retain continuity with the extracellular milieu. (C and D) Formation of conjugates between dendritic cells and T cells triggers actin-dependent polarization of virion conduits toward the contact zone with the T cell, resulting in contact of dendritic cell-associated viruses with CD4-rich filopodia/microvilli originating from the T cell. (E and F) Virus transfer from virion conduits to the T cell and diffusion along the surface of the T-cell membrane, likely driven by the active participation of cytoskeletal elements. The color burst in E schematically marks the proposed contacts at the dendritic cell-virus (cyan) and T-cell–virus (yellow) interface.

3D contacts between a dendritic cell and T cell at the synapse visualized with IA-SEM imaging. (A–C) Envelopment of the T cell by sheet-like membrane protrusions from the dendritic cell (Movies S1 and S2). The T-cells (numbered 1, 2, and 3) are colored yellow, and the dendritic cell is shown in light gray. Single-slice (A) and 3D-volume slabs before (B) and after (C) computational removal of the T cells labeled 2 and 3 from the imaged volume are shown. (D) Expanded view of C distinguishing the extensive membrane sheet encasement (magenta) and the localized filopodial interdigitations (green) at the region of cell-cell contact emanating from the dendritic cell. A schematic version of the contact is shown to the right. (E–J) Projection electron microscopic images from thin sections illustrating that every T cell is encased by membranes originating from the dendritic cell, with synapses visible in some instances. (Scale bars: 3 μm.)

Projection TEM images of synapses formed between virus-pulsed dendritic cells (DC) and T cells (T) preincubated with blocking antibodies. (A) Treatment with anti-CD4 antibody M-T477 results in virions polarized toward the contact region with the T cell but with viruses predominantly positioned on the dendritic cell and only rarely on the T cell. (B) Treatment with anti-CCR5 antibody 3A9 results in virus distribution similar to that seen in untreated cells (Fig. S4), with viruses present on both cell surfaces at the synapse (red circles). (Scale bars: 2 μm.)