ABSTRACT The mechanism of B cell–antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells.

Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune

complexes by a complement receptor–dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore,

cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an

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support SIMILAR CONTENT BEING VIEWED BY OTHERS DYNAMIC ENCOUNTERS WITH RED BLOOD CELLS TRIGGER SPLENIC MARGINAL ZONE B CELL RETENTION AND FUNCTION Article Open access 04 December 2023

SPATIAL MAPPING OF INNATE LYMPHOID CELLS IN HUMAN LYMPHOID TISSUES AND LYMPHOMA AT SINGLE-CELL RESOLUTION Article Open access 15 May 2025 LONG-TERM RETENTION OF ANTIGENS IN GERMINAL CENTERS

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M. Krummel for help with the two-photon microscope; F. Schaufele (Diabetes Endocrinology Research Center Imaging Core) for help with the confocal microscope; C. Allen for advice; T. Gerdes

and M. Wabl (University of California, San Francisco) for QM mice, J. Atkinson and X. Wu (University of Washington) for _Cr2__−/−_ mice; T. Kinoshita (Osaka University) and S. Boackle

(University of Colorado Health Sciences Center) for the 8C12 hybridoma; G. Kraal (Vrije University Medical Center) for the MOMA-1 hybridoma; M. Cooper (University of Alabama at Birmingham)

for monoclonal antibody BP-3; and G. Cinamon and L. Shiow for comments on the manuscript. Supported by the National Health and Medical Research Council and American Australian Association

(T.G.P.), the Cancer Research Institute (I.G.), the Howard Hughes Medical Institute (J.G.C.), the National Institutes of Health (AI45073 and AI40098) and a Sandler New Technology Award.

AUTHOR INFORMATION Author notes * Takaharu Okada Present address: Present address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto

University, Katsura Campus, Nishikyo-ku, Kyoto, Japan., AUTHORS AND AFFILIATIONS * Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California,

San Francisco, 94143, California, USA Tri Giang Phan, Irina Grigorova, Takaharu Okada & Jason G Cyster Authors * Tri Giang Phan View author publications You can also search for this

author inPubMed Google Scholar * Irina Grigorova View author publications You can also search for this author inPubMed Google Scholar * Takaharu Okada View author publications You can also

search for this author inPubMed Google Scholar * Jason G Cyster View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.G.P. and J.G.C. designed

and conceptualized the research; T.G.P. did the experiments; I.G. and T.O. assisted with the two-photon microscopy; and T.G.P., I.G. and J.G.C. analyzed the data and prepared the manuscript.

CORRESPONDING AUTHOR Correspondence to Jason G Cyster. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY

TEXT AND FIGURES Supplementary Figure 1 (PDF 1687 kb) SUPPLEMENTARY MOVIE 1 Intravital imaging of IC capture in the SCS. Time-lapse image sequence of 10 μm z-projection shows capture of

PE-ICs by cells lining the subcapsular sinus (annotated SCS). The SCS is visible between the collagen fibers (blue; second harmonic) of the capsule and the BP-3+ follicular stromal network

(green). PE-ICs were generated in vivo by subcutaneous injection of PE into a mouse that had previously received anti-PE IgG. Within minutes large PE-ICs (red) drain to the lymph node and

are visible flowing and accumulating on cells lining the SCS. The autofluorescent macrophage (labeled Mφ in the first few frames) protrudes into the lumen and is seen to rapidly accumulate

PE-ICs on its surface. PE-ICs can also be seen accumulating along a process of the macrophage (arrowhead). B cells (cyan) are not associated with any PE signal at this early time. Elapsed

time is shown as hh:mm:ss from the time of PE injection. Data are representative of three independent experiments. (MPG 3379 kb) SUPPLEMENTARY MOVIE 2 Real-time imaging of B cell migration

and interaction with SCS macrophages. Time-lapse image sequence of 20 μm z-projection from an inguinal lymph node explant 3 h after PE injection. By now the subcapsular space is coated with

PE-ICs (red) captured on SCS macrophages. B cells (cyan) continuously migrate to the SCS and interact with macrophages some of which are autofluorescent in the green channel. Capsule and SCS

associated collagen fibers appear blue (second harmonic). Elapsed time is shown as hh:mm:ss from the time of PE injection. Data are representative of three independent experiments. (MPG

6491 kb) SUPPLEMENTARY MOVIE 3 Real-time imaging of B cells capturing ICs in the subcapsular region. Time-lapse image sequence of 20 μm z-projection from an inguinal lymph node explant 3 h

after PE injection focusing on the subcapsular space which is coated with PE-ICs (red) captured on SCS macrophages. B cells (cyan) interact with SCS macrophages and can be seen to emerge

with associated PE signal on their surface (arrowheads). At one point a B cell (asterisk) approaches and captures a large depot of PE-ICs (open circle) presumably from the nearby SCS

macrophage (Mφ) and is depicted in Fig. 3c. Capsule and SCS associated collagen fibers appear blue (second harmonic). Elapsed time is shown as hh:mm:ss from the time of PE injection. Data is

representative of three independent experiments. (MPG 9500 kb) SUPPLEMENTARY MOVIE 4 Real-time imaging of B cells transporting PE-ICs into the follicle. Time-lapse image sequence of 20 μm

z-projection from an inguinal lymph node explant 4 h after PE injection. B cells (cyan) are highly motile and have dendritic extensions. The PE-ICs (red) are largely localized to the uropod

in the trailing edge of the B cells. The track of several B cells carrying PE-ICs are annotated with dots. Yellow dots shows the track of the B cell depicted in Fig. 3d. Elapsed time is

shown as hh:mm:ss from the time of PE injection. Data are representative of three independent experiments. (MPG 6465 kb) SUPPLEMENTARY MOVIE 5 Real-time imaging showing B cell expression of

complement receptors is required to capture PE-ICs. Time-lapse image sequence of 20 μm zprojection from inguinal lymph node explants 3 h after PE injection. CFSE-labeled (green) Cr1-,

Cr2-deficient (left panel) and wild-type B cells (right panel) in the subcapsular region are shown side-by-side for comparison. Capsule and SCS associated collagen fibers appear blue (second

harmonic). A wild-type B cell is highlighted (yellow arrow, right panel) that is in intimate contact with a macrophage for a prolonged period before emerging with PE-ICs on its surface.

Cr1-, Cr2-deficient B cells that encounter macrophages (highlighted with white arrows, left panel) are not seen to emerge with PE-IC. Note the dendritic morphology of migrating B cells even

in the absence of Cr1 and Cr2. ICs on B cells appear yellow due to co-localization of red and green. Elapsed time is shown as hh:mm:ss from the time of PE injection. Data are representative

of two independent experiments. (MPG 9615 kb) SUPPLEMENTARY MOVIE 6 Real-time imaging of B cells capturing and transporting cognate antigen coated with ICs. Time-lapse image sequence of 20

μm z-projection from inguinal lymph node explants showing efficient capture (left panel) and transport (right panel) of NP-PE ICs by cognate CFSE-labeled QM B cells (green) compared to

polyclonal CFP+ B cells (cyan). In the left panel a QM B cell (yellow arrowhead) can be seen scanning along the NP-PE IC coated process of a nearby macrophage (Mφ). Subsequently it rips off

some ICs and migrates away (white arrowhead) with NP-PE ICs capped at its tail. In the right panel several QM B cells can be seen migrating with large ICs capped at their tail. The QM B cell

tracked with yellow dots is depicted in Fig. 5c. Note the morphology of cognate QM B cells in contrast to polyclonal CFP+ B cells. Elapsed time is shown as hh:mm:ss from the time of NP-PE

injection. Data are representative of two independent experiments. (MPG 9654 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Phan, T., Grigorova, I.,

Okada, T. _et al._ Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. _Nat Immunol_ 8, 992–1000 (2007). https://doi.org/10.1038/ni1494

Download citation * Received: 29 May 2007 * Accepted: 25 June 2007 * Published: 29 July 2007 * Issue Date: September 2007 * DOI: https://doi.org/10.1038/ni1494 SHARE THIS ARTICLE Anyone you

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