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This item has the following additional materials available:
Movie 2D. Quick Time Movie (7187 KB)
Molecular accumulations at the interface of T cells and
fibroblasts are immunological synapses.
MCC:GFP cells and AD10 T cells were incubated for 30 minutes before
fixation and staining. With anti-PKCq (red)
and anti-phosphotyrosine (blue) Movie shows a 3D reconstruction of the
APC with 2 associated T cells. The redistributed MCC:I-Ek:GFP
colocalizes with PKCq (red) and
phosphotyrosine (blue) generating the white color at the interface.
Images taken at 600X.
Movie 2D supplement. Quick Time Movie (1749 KB)
Movie of the z axis data used to create movie 2D.
Movie 2J. Quick Time Movie (208 KB)
3Dimensional rotation of the accumulated molecules at the interface of
the APC and right T cell in movie 2D showing the colocalization of
MCC:I-Ek:GFP, anti-PKCq and
anti-phosphotyrosine.
Movie 3A. Quick Time Movie (1268 KB)
Small MHC clusters appear at the T-APC interface that intensify and coalesce to form a mature immunological synapse.
The movie contains a composite overlay of GFP images (in green) with
DIC images (pseudocolored blue). Small MHC clusters appear at the T-APC
interface that intensify and ultimately coalesce to form a mature
immunological synapse. The total duration of the movie is 16 minutes;
it begins 4 minutes before the first image in figure 3A (Labeled time
0) and concluded 1 minute after the last image in figure 3A. The image
capture interval was 12 seconds.
Movie 3B. Quick Time Movie (8166 KB)
Movement of the AD10 T cell across the surface of the
APC is associated with the movement of the mature immunological
synapse.
GFP (green) image is overlaid on DIC (blue) image. T cell interacts
with APC and over a period of 5 minutes, an immunological synapse
forms. The T cell then reinitiates locomotion across the APC and the
synapse moves as well. In 13 minutes, the synapse has moved a total of
24 µm. The image frequency is 10 seconds and the total time of
this movie is 19 minutes.
Movies 3C. Quick Time Movie (4776 KB)
Immunological synapses formed are
antigen-specific.
When MCC:GFP cells are pulsed with Hb peptide, the Hb-specific T cells
recognize antigen and form stable conjugates morphologically identical
to AD10 -MCC:GFP conjugates. No redistribution of
MCC:I-Ek:GFP is observed. Movie represents 40 minutes with
an image frequency is 12 seconds.
Movie 4. Quick Time Movie (7624 KB)
Ca++ Flux Within the T Cell Precedes Synapse
Formation
Three Fura-2 loaded AD10 T cells make contact with the APC and flux
Ca++. Ca++ flux occurs before, or concurrent with, the first visible
MHC clusters and several minutes before mature immunological synapse
formation. The first part of the movie shows Fura-2 imaged in green
(340 nm) and red (380 nm), which are overlaid to form a yellow color in
the non-activated T cells. As intracellular Ca++ concentrations
increase, the 340 nm signal increases and the 380 signal decreases
resulting in a color shift from yellow to green. The GFP on the APC is
pseudocolored blue and synapses are visible as bright blue regions at
the T - APC interface. To better visualize synapse formation in the
second portion of the movie, fluorescent (green) and DIC images (blue)
are overlaid. Images were obtained at 600X. The image interval is 12
seconds and the total duration of the movie is 14 minutes.
Movie 5D. Quick Time Movie (3185 KB)
3D interface of AD10 T cells and MCC:GFP cells
after treatment of APC with 25 µg/ml human IgG. Note the large
central GFP cluster.
Movie 5H. Quick Time Movie (604 KB)
3D interface of AD10 T cells and MCC:GFP cells
after treatment of APC with 25 µg/ml CTLA-4Ig. The size and
intensity are significantly reduced compared to IgG control (movie 5D).
The morphology of the synapse is also clearly altered.
Movie 5L. Quick Time Movie (594 KB)
3D interface of AD10 T cells and MCC:GFP cells
after treatment of APC with 25 µg/ml anti-ICAM-1. The size and
intensity are significantly reduced compared to IgG control (movie 5D).
The morphology of the synapse is also clearly altered.
Movie 5P. Quick Time Movie (515 KB)
3D interface of AD10 T cells and MCC:GFP cells
after treatment of APC with 25 µg/ml anti-I-Ek. The
size and intensity are significantly reduced compared to IgG control
(movie 5D). The morphology of the synapse is also clearly altered.
Prepared by: the Data Supplement Manager
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