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Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells

Abstract

Lymph nodes prevent the systemic dissemination of pathogens such as viruses that infect peripheral tissues after penetrating the body’s surface barriers. They are also the staging ground of adaptive immune responses to pathogen-derived antigens1,2. It is unclear how virus particles are cleared from afferent lymph and presented to cognate B cells to induce antibody responses. Here we identify a population of CD11b+CD169+MHCII+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of lymph nodes that capture viral particles within minutes after subcutaneous injection. Macrophages in the SCS translocated surface-bound viral particles across the SCS floor and presented them to migrating B cells in the underlying follicles. Selective depletion of these macrophages compromised local viral retention, exacerbated viraemia of the host, and impaired local B-cell activation. These findings indicate that CD169+ macrophages have a dual physiological function. They act as innate ‘flypaper’ by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph–tissue interface that facilitate the recognition of particulate antigens by B cells and initiate humoral immune responses.

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Figure 1: Capture of lymph-borne VSV by SCS macrophages.
Figure 2: Macrophage-mediated transfer of lymph-borne VSV across the SCS floor alters virus-specific B cell behaviour.
Figure 3: SCS macrophages are required for early activation of VSV-specific B cells in lymph nodes.

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Acknowledgements

We thank G. Cheng, M. Flynn and D. Baumjohann for technical support; R. M. Zinkernagel and H. Hengartner for providing VI10YEN mice; A. Wagers for providing Act(EGFP) mice; M. Ericsson and E. Benecchi for expert support in electron microscopy studies; S. Behnke for immunohistochemistry; and D. Cureton for help and advice with VSV preparations. This work was supported by grants from the NIH-NIAID (to U.H.v.A.), a Pilot and Feasibility Grant from the Harvard Skin Disease Research Center (to T.J. and U.H.v.A.), a stipend from the Swiss National Science Foundation (to T.J.) and a NIH T32 Training Grant in Hematology (to E.A.M.).

Author Contributions T.J. and U.H.v.A. designed the study; T.J., E.A.M., M.I., S.M. and P.A.L. performed experiments; T.J., E.A.M., M.I. and S.M. collected and analysed data; M.B., K.F., N.C.D.P., D.M.S., N.v.R. and S.P.W. provided reagents and mice; T.J., E.A.M., M.I. and U.H.v.A. wrote the manuscript; S.M., K.F., S.E.H., T.M. and S.P.W. gave technical support and conceptual advice.

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Correspondence to Ulrich H. von Andrian.

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Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-10 with Legends and Legends to Supplementary Movies 1-8. (PDF 22824 kb)

Supplementary Movie 1

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. (MOV 2970 kb)

Supplementary Movie 2

This file contains Supplementary Movie 2 which shows entry of VSV particles into the SCS of a popliteal LN in a C57BL/6→Act(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/6→Act(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. (MOV 3826 kb)

Supplementary Movie 3

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. (MOV 2479 kb)

Supplementary Movie 4

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. (MOV 3825 kb)

Supplementary Movie 5

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. (MOV 4021 kb)

Supplementary Movie 6

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. (MOV 3832 kb)

Supplementary Movie 7

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. (MOV 3098 kb)

Supplementary Movie 8

This file contains Supplementary Movie 8 which shows VSV-IND is internalized by VI10YEN B cells and colocalizes with MHC-class II in endocytic compartments. 18 h following intravenous transfer of 5-6x106 purified VI10YENxMHCII(EGFP) B cells (green), 20 mg AlexaFluor-568 labeled UV-VSV-IND (red) was injected s.c. into the hind footpad of recipient mice. LNs were removed 30 minutes after injection of virus. 30mm cryosections were analyzed by confocal microscopy. Movie is Z stack of 25 sections (z increment of 0.5 mm) at a pixel density of 512x512. (MOV 979 kb)

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Junt, T., Moseman, E., Iannacone, M. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007). https://doi.org/10.1038/nature06287

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