Supplemental Data
Supplemental Data for
Egen et al., Immunity 28, pp. 271-284
- Document S1. Seven Figures
- Movie S1. Rapid Association of Blood-Borne
BCG with Kupffer Cells Observed in MHCII-EGFP Mice
Two-dimensional time-lapse image series from MHCII-EGFP mice
that were infected with 1 × 107 BCG-RFP bacteria through an i.v. catheter 1 min after beginning imaging. EGFP-expressing cells
are shown in green, BCG-RFP fluorescence is shown in red, and a fluorescent blood tracer is shown in blue. Scale bar represents
20 μm. Playback speed is 30×.
- Movie S2. Rapid Association of Blood-Borne BCG with Kupffer
Cells Observed in LysM-EGFP Mice
Two-dimensional time-lapse image series from LysM-EGFP mice that were infected
with 1 × 107 BCG-RFP bacteria through an i.v. catheter 1 min after beginning imaging. For details, see legend for Movie S1.
- Movie S3. Kupffer Cell-Associated BCG Appear to Be Located in an Intracellular Compartment
A
LysM-EGFP mouse that was being imaged by hepatic IVM was infected with BCG-RFP bacteria through an i.v. catheter. 15 min later,
a high-resolution 3-dimensional stack was captured of a Kupffer cell (green) lining the sinusoidal vessels (blue) and associated
with BCG-RFP (red). The movie is a volume rendering of the image stack showing first a projection image of the entire data
set, then a volumetric surface rendering of the Kupffer cell and bacteria, and finally a cut-away section from the surface
of the Kupffer cell revealing the intracellular bacteria.
- Movie S4. Neutrophils Are Recruited to
the Liver Sinusoids but Do Not Directly Capture Mycobacteria
Four-dimensional image series from the liver of
a LysM-EGFP mouse that had been infected with BCG-RFP bacteria i.v. 3 hr earlier. The data set is played twice during the
movie, once with the blood tracer fluorescence visible (blue) and once with this channel removed. Notice the large number
of Kupffer cells (static cells expressing low levels of EGFP [green]) containing fluorescent BCG (red) and the absence of
interactions between mycobacteria and neutrophils (motile cells expressing high levels of EGFP). Scale bar represents 80 μm.
Playback speed is 300×.
- Movie S5. EGFP-Expressing, BCG-Infected Cells Are Recruited into Granulomas
A
high-resolution 3-dimensional image of a granuloma from the liver of a LysM-EGFP bone-marrow chimera that had been infected
with BCG-RFP bacteria for 3 weeks. EGFP-expressing cells are shown in green, F4/80 expressing cells are shown in red, and
BCG-RFP are shown in white. The movie is a volume rendering of the image stack showing first a projection image of the entire
data set, then a volumetric surface rendering of the Kupffer cell and bacteria, and finally a cut-away section from the surface
of the Kupffer cell revealing the intracellular bacteria.
- Movie S6. Limited Macrophage and Mycobacterial
Dynamics during Granuloma Development
4-dimensional image series from the liver of a LysM-EGFP mouse that had
been infected with BCG-RFP bacteria for 1 week. Dimensions of the imaging field are 180 × 180 × 46 μm. EGFP-expressing
cells are shown in green and BCG-RFP are shown in red. Playback speed is 300×.
- Movie S7. Limited
Macrophage and Mycobacterial Dynamics during Granuloma Development
4-dimensional image series from the liver
of a LysM-EGFP mouse that had been infected with BCG-RFP bacteria for 2 weeks. Dimensions of the imaging field are 180 × 180
× 60 μm. For details, see legend to Movie S6.
- Movie S8. Limited Macrophage and Mycobacterial
Dynamics during Granuloma Development
4-dimensional image series from the liver of a LysM-EGFP mouse that had
been infected with BCG-RFP bacteria for 3 weeks. Dimensions of the imaging field are 180 × 180 × 40 μm. For details,
see legend to Movie S6.
- Movie S9. Limited Macrophage and Mycobacterial Dynamics during Granuloma
Development
A low-magnification 4-dimensional image series from the liver of a LysM-EGFP mouse that had been
infected with BCG-RFP bacteria for 3 weeks. EGFP-expressing cells are shown in green and BCG-RFP are shown in white. Scale
bar represents 80 μm. Playback speed is 300×.
- Movie S10. Limited Motility of Macrophages
inside Granulomas
4-dimensional image series from the liver of a LysM-EGFP mouse that had been infected with
BCG-RFP bacteria for 3 weeks. EGFP-expressing cells are shown in green. The left panel shows the raw imaging data of a granuloma
while the right panel shows the same granuloma after subtraction of a mean intensity time projection image from each time
point. This method is used to preferentially remove fluorescence intensity that remains constant over time (see Figure S7).
Dimensions of the imaging field are 135 × 135 × 30 μm. Playback speed is 300×.
- Movie S11.
Rapid Motility but Restricted Migration of T Cells inside Mature Granulomas
4-dimensional image series from a
RAG1-deficient mouse that had been reconstituted with EGFP-expressing CD4+ T cells and infected with BCG-RFP bacteria 3 weeks
earlier. EGFP-expressing cells are shown in green and sinusoids are shown in white. Scale bar represents 40 μm. Playback
speed is 300×.
- Movie S12. Granuloma-Associated T Cells Swarm around a Core of Mycobacteria-Infected
Cells
4-dimensional image series from a RAG1-deficient mouse that had been reconstituted with EGFP-expressing
CD4+ T cells and infected with BCG-RFP bacteria 3 weeks earlier. EGFP-expressing cells are shown in green and BCG-RFP are
shown in white. Scale bar represents 40 μm. Playback speed is 300×.
- Movie S13. T Cell Division
Can Be Seen inside Mature Granuloma
4-dimensional image series from a RAG1-deficient mouse that had been reconstituted
with EGFP-expressing CD4 T cells and infected with BCG-RFP 3 weeks earlier. EGFP-expressing cells are shown in green and BCG-RFP
are shown in red. Movie starts by highlighting dividing cell. Scale bar represents 20 μm. Playback speed is 300×.
- Movie S14. Effector T Cells Are Rapidly Recruited to Established Granulomas in an Antigen-Independent Manner
4-dimensional
image series from a C57BL/6 mouse that had been infected with BCG 3 weeks earlier. In vitro stimulated, dye-labeled OT-II
TCR transgenic T cells were injected into the animal through an i.v. catheter 15 min after the start of imaging. T cells are
shown in red and sinusoids are shown in white. Dimensions of the imaging field are 234 × 240 × 30 μm. Playback speed
is 300×.
- Movie S15. Newly Recruited Granuloma-Associated T Cells Show Rapid Motility and Restricted
Migration
4-dimensional image series from a C57BL/6 mouse that had been infected with BCG 3 weeks earlier. Animals
received in vitro stimulated, dye-labeled OT-II TCR transgenic T cells 12 hr prior to imaging. EGFP-expressing cells are shown
in green and T cells are shown in red. Dimensions of the imaging field are 230 × 230 × 33 μm. Playback speed is 300×.
- Movie S16. T Cells Must Squeeze through a Dense Macrophage Network when Migrating inside a Granuloma
4-dimensional
image series from the center of a granuloma in the liver of a LysM-EGFP mouse that had been infected with BCG 3 weeks earlier.
Animals received in vitro stimulated, dye-labeled OT-II TCR transgenic T cells 12 hr prior to imaging. The left panel shows
EGFP-expressing cells in green and the right panel shows an overlay of EGFP fluorescence, OT-II T cells in red, and sinusoids
in white. In the second half of the movie, a region of the granuloma is expanded and the image series is repeated, running
in slow motion at several key points during playback. Arrows point to examples of transferred T cells migrating through the
dense macrophage network. Notice the displacement of green fluorescence as the T cell migrate in between macrophages. Scale
bar represents 30 μm. Playback speed is 300× except during slow-motion sequences.
- Movie
S17. Restricted T Cell Migration inside Granulomas Is Defined by a Macrophage-Delineated Border
4-dimensional
image series from a LysM-EGFP mouse that had been infected with BCG 3 weeks earlier. Animals received in vitro stimulated,
dye-labeled OT-II TCR transgenic T cells 12 hr prior to imaging. EGFP-expressing cells are shown in green and T cells are
shown in red. Sinusoids are briefly shown in white before the channel is removed to more clearly depict the macrophage-T cell
relationship. Dimensions of the imaging field are 150 × 162 × 56 μm. Playback speed is 300×.
- Movie
S18. T Cells Appear to Migrate along Macrophage Cell Bodies at the Granuloma Periphery
4-dimensional image series
from a LysM-EGFP mouse that had been infected with BCG 3 weeks earlier. Animals received in vitro stimulated, dye-labeled
OT-II TCR transgenic T cells 12 hr prior to imaging. EGFP-expressing cells are shown in green and T cells are shown in red.
The track of an individual T cell approaching the granuloma border is shown in blue. Dimensions of the imaging field are 63
× 63 × 42 μm. Playback speed is 180×.
- Movie S19. T Cells Appear to Migrate along Macrophage
Cell Bodies at the Granuloma Periphery
Similar to Movie S18. EGFP-expressing cells are shown in green, T cells
are shown in red, and sinusoids are shown in white. Tracks of individual T cells approaching the granuloma border are shown
in blue. Dimensions of the imaging field are 100 × 100 × 28 μm. Playback speed is 180×.
- Movie
S20. T Cells Are Recruited to Immature Granulomas Where They Migrate in Close Apposition to Macrophages
4-dimensional
image series from a LysM-EGFP mouse that had been infected with BCG 12 days earlier. Animals received in vitro stimulated
OT-II TCR transgenic T cells 12 hr prior to imaging. EGFP-expressing cells are shown in green, T cells are shown in red, and
sinusoids are shown in white. Dimensions of the imaging field are 179 × 175 × 54 μm. Playback speed is 300×.
