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Blood, Vol. 104, Issue 9, 2801-2809, November 1, 2004
A spectrum of biophysical interaction modes between T cells and different antigen-presenting cells during priming in 3-D collagen and in vivo
Blood Gunzer et al.
104: 2801
Supplemental materials for: Gunzer et al, Vol 104, Issue 9, 2801-2809
Files in this Data Supplement:
- Movie 1. Migration of a T-B pair in 3-D collagen (MOV, 4,810 KB)
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A pair made up of a T cell and a naive B cell (green) is seen migrating in a curve within a 3-D collagen matrix (red box). The B cell forms a round head on top of the highly motile T cell. Time is 120 seconds per frame and 140 minutes total observation. The movie was taken 5 hours after the onset of the culture. Note: In all movie sequences, B cells, DCs, and macrophages were loaded with 100 µg/mL OVA 323-339 peptide. T cells were always freshly prepared.
- Movie 2. Calcium flux in T cells associated with B cells (MOV, 3,955 KB)
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A T cell–B cell pair (T cell is rainbow-colored; B cell, red) migrates through the gel. The color signal is due to fluorescence of the Ca2+ labeling dye, Fluo4 (Molecular Probes, Leiden. The Netherlands), indicating Ca2+ signaling within the T cell upon the cognate contact and during migration. The calcium flux in the T cell demonstrates that the observed contacts between T cells and B cells induce functional T-cell activation. Time is 60 seconds per frame and approximately 30 minutes total observation. The movie was taken 2 hours after the onset of the culture.
- Movie 3. Short-lived contacts of T cells with DC (MOV, 2,223 KB)
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T cells interacting with several peptide-loaded DCs within a collagen gel. Note that the contacts are very dynamic and short (1 frame is approximately 80 seconds). Some cells can be seen making several contacts with the same or another DC. Time is 80 seconds per frame and 5 hours total observation. The movie was taken 25 hours after the onset of the culture.
- Movie 4. DC use their cytoskeleton to actively grab T cells (MOV, 3,573 KB)
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Two sequences show a DC beginning an interaction with a T cell. The first sequence shows a migratory T cell being engaged by the DC (red arrowhead); the second sequence, a T cell that is nonmotile before contact (red box). Note how the DC makes multiple contacts with individual pseudopods before enwrapping and finally grabbing the entire T cell. The T cell keeps migrating over the DC?s surface and makes contact with a second DC above it (sequence 1) or is dragged away by the migrating DC (sequence 2). Time is 21 minutes for sequence 1 and 43 minutes for sequence 2. The first movie was taken 35 hours after the onset of the culture; the second, 1 hour after onset.
- Movie 5. T-B pairs are very stable (MOV, 2,398 KB)
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A cognate T cell–B cell pair is shown (red box) migrating intensively while maintaining a permanent contact for more than 17 hours, until the T cell releases and migrates away and the B cell remains. Due to dye bleaching, such long observations are not possible with carboxyfluorescein diacetate (CFDA, Molecular Probes)-staining of B cells. However, the observation of many CFDA-stained T cell–B cell pairs has enabled us to clearly identify T cell–B cell interactions by using morphologic criteria. Real time of the experiment is indicated by the white numbers (hours and minutes). The movie starts 24 hours after the onset of the culture.
- Movie 6. B cells are passively pushed during T cell contact (MOV, 4,745 KB)
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An established T cell–B cell pair (in the red box, with the T cell unstained and the B cell green) migrates through the collagen matrix, moved forward by the motility of the T cell. After approximately 20 minutes the pair breaks up (the movie pauses for 2 seconds), and the B cell then develops autonomous effective locomotion. After 160 minutes another T cell–B cell pair comes along and the T cell exchanges its previously contacted B cell for the new one (the movie pauses for 2 seconds). When the previously motile B cell forms a contact to the new T cell, all autonomous migration by the B cell stops and the B cell is passively transported by the motile T cell again. Time is 120 seconds per frame, and approximately 200 minutes total observation. The movie was taken 30 minutes after the onset of the culture.
- Movie 7. Extreme motility of T-B pairs in 3-D collagen (MOV, 1,624 KB)
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A T cell–B cell pair (shown by the white arrow, then by the red box) migrates extremely vigorously through the gel, constantly maintaining tight cell-cell contact. Time is 60 seconds per frame and approximately 116 minutes total observation. The movie was taken 48 hours after the onset of the culture.
- Movie 8. Stable contact plane in a T-B pair (MOV, 4,503 KB)
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A T cell–B cell pair is shown in higher resolution. Note that despite effective migration, the contact plane remains relatively stable and fixed, while the T cell constantly produces membrane ruffles around the contact site. Time is 15 seconds per frame and approximately 80 minutes total observation. The movie was taken 72 hours after the onset of the culture.
- Movie 9. Pronounced cytoskeletal activity of activated B cells (MOV, 2,032 KB)
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A close-up view of migrating activated B cells (green). Shortly before the end of the movie, a pairing of an activated B cell and an unstained T cell comes into view, the T cell being dragged by the activated B cell. Time is 120 seconds per frame and approximately 220 minutes total observation. The movie was taken 30 minutes after the onset of the culture.
- Movie 10. Activated B cells use their cytoskeleton to actively grab T cells (MOV, 1,657 KB)
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An activated B cell is seen establishing a contact with a resting T cell. Note how the activated B cell stretches membrane protrusions towards the T cell, circumvents it after contact formation, and subsequently drags the T cell away. Time is 120 seconds per frame and approximately 140 minutes total observation. The movie was taken 30 minutes after the onset of the culture.
- Movie 11. T-B pairs in a lymph node of a living mouse (MOV, 5,022 KB)
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Intravital imaging of an established T cell–B cell pair migrating at the interface of the T-cell zone and a B-cell follicle of the inguinal lymph node in a living Balb/c mouse. The T cell is stained red with Celltracker orange (Molecular Probes), and the B cell is stained green with CFDA (Molecular Probes). Time is 45 seconds per frame and approximately 50 minutes total observation. The movie was taken 4 hours after injection of T cells.
- Movie 12. A single T-B pair within a lymph node in high resolution (MOV, 2,501 KB)
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Intravital imaging of multiple T cell–B cell pairs migrating at the interface of the T-cell zone and a B-cell follicle and within the follicle of the inguinal lymph node in a living Balb/c mouse. T cells are stained red with Celltracker orange, and the B cells are stained green with CFSE. During the movie, the focus slightly shifts upward (due to movements of the animal and blood flow) and reveals that the T-cell zone extends over the ball-like B-cell follicle. The intensity of the red channel has been increased to reveal T cells not in full focus, as well as the matrix structure of the node. Note the extensive autonomous migration of B cells not in contact with a T cell. Time is 60 seconds per frame and approximately 30 minutes total observation. The movie was taken 7 hours after injection of T cells.
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