von Andrian Lab Videos
 

 

SUBCAPSULAR SINUS MACROPHGES IN LYMPH NODES CLEAR LYMPH-BORNE VIRUSES AND PRESENT THEM TO ANTIVIRAL B CELLS

Nature Supplementary Movie s1.mov (2.9MB)

This file contains Supplementary Movie 1 which shows how, following footpad injection, VSV particles rapidly accumulate in a patchy pattern on the floor of the SCS of the draining popliteal LN.
20 μg AlexaFluor-488 labeled UV-irradiated VSV (green) were injected in 20 μl PBS into the hind footpad of an anesthetized mouse while the SCS and superficial parenchyma of the draining popliteal LN was recorded by MP-IVM. Z-stacks of 11 optical sections (z-increment 4 μm) were acquired every 15 seconds over 30 minutes at a pixel density of 256x256. This movie shows a maximum intensity projection, which displays at each pixel only the brightest value encountered along an axial viewing ray in each color channel. The second harmonic signals from collagen fibers in the LN capsule and the adventitia of the adjacent saphenous vein are shown in blue. Fluorescent VSV accumulated rapidly on the floor of the SCS, but did not enter the LN parenchyma. The counter in the upper left corner shows minutes and seconds after VSV injection. Scale bar corresponds to 100 μm.

 


Nature Supplementary Movie s2.mov (3.7MB)

This file contains Supplementary Movie 2 which shows entry of VSV particles into the SCS of a popliteal LN in a C57BL/6Act(EGFP) chimeric mouse. AlexaFluor-568 labeled UV-irradiated VSV particles (red) were injected into a hind footpad of an anesthetized mouse and recorded by MP-IVM as described in the legend to Suppl. Movie 1. The field of view chosen at a lateral aspect of the draining popliteal LN, with the parenchyma on the left, the SCS running vertically across the center and extranodal connective tissue on the right. Non-hematopoietic EGFP+ cells in C57BL/6Act(EGFP) chimeras, mostly sinus-lining lymphatic endothelial cells, are shown in green. Second harmonic signals from collagen fibers in the LN capsule and extranodal tissue are visible in blue. The counter in the upper left corner shows minutes and seconds after VSV injection. The scale bar corresponds to 50 μm.

 


Nature Supplementary Movie s3.mov (2.4MB)

This file contains Supplementary Movie 3 which shows sustained and selective retention of fluorescent VSV on the floor of the SCS. This recording was taken 3h after footpad injection of AlexaFluor-568 labeled UV-VSV (red) in the same preparation as shown in Suppl. Movie 2. The movie shows a projection through a z-stack of 15 optical sections (z-increment of 2 μm) at a pixel density of 512x512. The image is rotated ±20º around the y-axis. Note that the red viral deposits do not overlap with the green EGFP signal associated with non-hematopoietic cells. The scale bar corresponds to 50 μm.

 


Nature Supplementary Movie s4.mov (7.6MB)

This file contains Supplementary Movie 4 which shows that In the absence of virus VI10YENand polyclonal B cells have equivalent distributions and velocities throughout the popliteal LN. Purified, CMTMR labeled VI10YEN B cells (red) and CMAC labeled polyclonal B cells (blue) were transferred i.v. at 5-6x106 cells each into C57BL/6 recipients, 18 h prior to recording. A B cell follicle in the popliteal LN was continuously recorded by MP-IVM over 119 frames, at a frame-to-frame interval of 15 seconds, with Z stack of 11 sections (z increment of 4 mm) and a pixel density of 256x256. The counter in the upper left corner indicates minutes and seconds. Scale bar, 50 mm.

 


Nature Supplementary Movie s5.mov (3.9MB)

This file contains Supplementary Movie 5 which shows VI10YEN and wildtype B cells have equivalent distributions and velocities after introduction of VSV-NJ. Purified, CMTMR labeled VI10YEN B cells (red) and CMAC labeled polyclonal B cells (blue) were transferred i.v. at 5-6x106 cells each into C57BL/6 recipients, 18 h prior to recording. A B cell follicle in the popliteal LN was continuously recorded by MP-IVM over 119 frames, at a frame-to-frame interval of 15 seconds, with Z stack of 11 sections (z increment of 4 mm) and a pixel density of 256x256. White numbers in upper left corner indicate minutes and seconds, scale bar corresponds to 50 mm. The recipient mouse received a footpad injection of 20μg AlexaFluor-488 labeled VSV-NJ (green) 5 minutes prior to the start of the recording. This 3D time-lapse recording was taken from 5-30 min after VSV-NJ injection. The counter in the upper left corner indicates minutes and seconds. Scale bar, 50 μm.

 


Nature Supplementary Movie s6.mov (7.7MB)

This file contains Supplementary Movie 6 which shows VI10YEN B cells rapidly accumulate below and within the SCS floor after introduction of VSV-IND. The experimental protocol and image acquisition was as in Suppl. Movie 5, except the recipient mouse received a footpad injection of 20μg AlexaFluor-488 labeled VSV-IND (green) 5 minutes prior to the start of the recording. This 3D time-lapse recording was taken from 5-30 min after VSV-IND injection. The counter in the upper left corner indicates minutes and seconds. Scale bar, 50 μm.

 


Nature Supplementary Movie s7.mov (3.0 MB)

This file contains Supplementary Movie 7 which shows VSV-IND induces VI10YEN B cell arrest but not accumulation at the SCS in CLL-treated popliteal LNs. The experimental protocol and image acquisition was as in Suppl. Movie 5, but in mice that received a footpad injection of CLL 7 d prior to imaging. The recipient mouse received a footpad injection of 20μg Alexa-488 labeled VSV-IND (green) 5 minutes prior to the start of the recording. This 3D time-lapse recording was taken from 5-30 min after VSV-NJ injection. The counter in the upper right corner indicates minutes and seconds. Scale bar, 50 μm. Note that fluorescent VSV appears in the flowing lymph, but is not retained at the SCS floor. Only the rare VI10YEN cells in the SCS lumen have access to the lymph-borne virus as evidenced by acquisition of green fluorescence.

 


Nature Supplementary Movie s8.mov (980 KB)

 

This file contains Supplementary Movie 7 which shows VSV-IND induces VI10YEN B cell arrest but not accumulation at the SCS in CLL-treated popliteal LNs. The experimental protocol and image acquisition was as in Suppl. Movie 5, but in mice that received a footpad injection of CLL 7 d prior to imaging. The recipient mouse received a footpad injection of 20μg Alexa-488 labeled VSV-IND (green) 5 minutes prior to the start of the recording. This 3D time-lapse recording was taken from 5-30 min after VSV-NJ injection. The counter in the upper right corner indicates minutes and seconds. Scale bar, 50 μm. Note that fluorescent VSV appears in the flowing lymph, but is not retained at the SCS floor. Only the rare VI10YEN cells in the SCS lumen have access to the lymph-borne virus as evidenced by acquisition of green fluorescence.



For details on experimental methods & techniques see:
Junt, T., Moseman, E.A., Boes, M., Fink, K., Shayakhmetov, D.M., Mempel, T.R., Whelan, S.P. and von Andrian, U.H. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature, 2007 Oct 14; [Epub ahead of print] PMID: 17934446. [Full Text PDF]