Abstract
A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.
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Acknowledgements
We thank J. Nolting (Dana Farber Cancer Institute) for hemagglutinin–T cell antigen receptor–transgenic mice; M. Nussenzweig (Rockefeller University) for CD11c-eYFP mice; R. Steinman (Rockefeller University) for the anti-SIGN-R1 hybridoma; K. Rajewsky (Harvard Medical School) for B1.8 mice; W. Gerhard (Wistar Institute) for the anti-hemagglutinin hybridoma; A. Garcia-Sastre (Mount Sinai School of Medicine) for influenza A/PR/8; J. Jensenius (Aarhus University) and S. Thiel (Aarhus University) for anti-MBL and technical advice; T. Mempel for the use of microscopy facilities and technical assistance; and H. Leung, E. Marino, M. Ericsson, H. Kim and A. Gillmore for technical assistance. Supported by the US National Institutes of Health (5 R01 AI039246, 1 P01 AI078897 and 5 R01 AI067706 to M.C.C., RO1 GM62444 to M.J.C. and RO1 DK074500 to S.J.T.) and the Seventh Framework Programme of the European Union (Marie Curie International Outgoing Fellowship 220044 to S.F.G.).
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S.J.T. and M.C.C. directed the study, designed experiments, analyzed and interpreted results and wrote the manuscript; S.F.G., V.L.-K., M.P.K., L.A.P., S.E.D. and Y.-A.K. designed experiments, analyzed and interpreted results; M.J.C. prepared dendrimer; and L.M.-P. and S.G. contributed reagents and helped to interpret results.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Methods (PDF 3443 kb)
Supplementary Movie 1
Subcapsular sinus macrophages (SSM) and medullary macrophages (MM) bind influenza virus in the draining LN. WT mice were pretreated with CD169 (green) and F4/80 (blue) antibodies to label SCS and MM respectively. Mice then prepared for MP-IVM, whole LN imaged and fluorescently labeled PR8 (red) was injected at time = 0 min. PR8 localization in the LN was imaged by MP-IVM at 15 s intervals for 30 min. PR8 (red) enters the SCS area within 8 min p.i. and remains in the SCS at 30 min where it colocalizes with CD169+ SSM and F4/80+ MM. Results are representative of 6 independent experiments, n = 6 mice total. (AVI 3197 kb)
Supplementary Movie 2
Mannose dendrimer blocks binding of PR8 to SSM, but not MM. Mice pretreated with CD169 (green) and anti-CD35 (blue) Ab to label SSM and FDC in vivo. Mice were subsequently treated with 40 μg of dendrimer in the footpad and were prepared for whole LN MP-IVM. Images were recorded beginning at time of fluorescently labeled PR8 injection (red, t = 0) at 15 sec intervals for 30 min. This movie demonstrates that following pretreatment with dendrimer, PR8 (red) does not bind to CD169+ SSM, but instead drains to the LN medulla where it binds to MM. Movie for 30 min p.i. Results are representative of 4 independent experiments, n = 4 mice total. (AVI 2922 kb)
Supplementary Movie 3
Medullary CD11c-EYFP cells bind virus is SIGN-R1 dependent. MP-IVM recordings of the LN medulla of a CD11c-YFP mouse (left images) and a CD11c-EYFP mouse treated with mAb against SIGN-R1 (22D1) to transiently knockout (TKO) SIGN-R1 expression (right images). The medulla of the popliteal LN were examined by MP-IVM beginning at t = 0 when PR8 (red) was injected into the footpad. Images were acquired every 30 sec for 120 min. Virus up take by CD11c-EYFP+ cells was substantially reduced in SIGN-R1 TKO mice, indicating that SIGN-R1 is crucial for CD11c-EYFP+ cells to bind virus. Results are representative of 4 independent experiments, n = 8 mice total. (MOV 12283 kb)
Supplementary Movie 4A
CD11c-EYFP cells bind PR8 and move to the FDC region. CD11c-EYFP mice were pretreated with anti-CD35 to label follicles and prepared for MP-IVM. An area of the LN was chosen for further analysis where the medullary region and FDC region occurred in one field of the MP (medullary region = top left, FDC region = bottom right). A baseline recording was performed for 60 min (a) and then fluorescently labeled PR8 (red) was injected into the footpad. MP-IVM recordings were continued for a further 60 min (b,c). 50 randomly chosen CD11c-EYFP+ cells were tracked prior to injection of PR8 (a) and then 50 randomly chosen CD11c-EYFP+ cells that captured PR8 (b) and 50 randomly chosen CD11c-EYFP+ cells which did not capture PR8 were also tracked (c). Results are representative of 4 independent experiments, n = 4 mice total. (AVI 2615 kb)
Supplementary Movie 4B
CD11c-EYFP cells bind PR8 and move to the FDC region. CD11c-EYFP mice were pretreated with anti-CD35 to label follicles and prepared for MP-IVM. An area of the LN was chosen for further analysis where the medullary region and FDC region occurred in one field of the MP (medullary region = top left, FDC region = bottom right). A baseline recording was performed for 60 min (a) and then fluorescently labeled PR8 (red) was injected into the footpad. MP-IVM recordings were continued for a further 60 min (b,c). 50 randomly chosen CD11c-EYFP+ cells were tracked prior to injection of PR8 (a) and then 50 randomly chosen CD11c-EYFP+ cells that captured PR8 (b) and 50 randomly chosen CD11c-EYFP+ cells which did not capture PR8 were also tracked (c). Results are representative of 4 independent experiments, n = 4 mice total. (AVI 3213 kb)
Supplementary Movie 4C
CD11c-EYFP cells bind PR8 and move to the FDC region. CD11c-EYFP mice were pretreated with anti-CD35 to label follicles and prepared for MP-IVM. An area of the LN was chosen for further analysis where the medullary region and FDC region occurred in one field of the MP (medullary region = top left, FDC region = bottom right). A baseline recording was performed for 60 min (a) and then fluorescently labeled PR8 (red) was injected into the footpad. MP-IVM recordings were continued for a further 60 min (b,c). 50 randomly chosen CD11c-EYFP+ cells were tracked prior to injection of PR8 (a) and then 50 randomly chosen CD11c-EYFP+ cells that captured PR8 (b) and 50 randomly chosen CD11c-EYFP+ cells which did not capture PR8 were also tracked (c). Results are representative of 4 independent experiments, n = 4 mice total. (AVI 3405 kb)
Supplementary Movie 5
Virus binding in the popliteal LN is MBL and SIGN-R1 dependent. MBL-deficient mice pretreated with SIGN-R1 were pretreated with MOMA-1 (green), F4/80 and 8C12 (blue) to label the SCS, medullary region and follicles, respectively. Virus localization in the LN was visualized using MP-IVM following injection of fluorescently labeled PR8 (red) into the footpad, time = 0 min, recordings made every 15 s for 60 min. Negligible virus is observed in the SCS or in the medullary region in MBL deficient mice pretreated with SIGN-R1 blocking antibodies. Results are representative of 3 independent experiments, n = 3 mice total. (AVI 5016 kb)
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Gonzalez, S., Lukacs-Kornek, V., Kuligowski, M. et al. Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes. Nat Immunol 11, 427–434 (2010). https://doi.org/10.1038/ni.1856
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DOI: https://doi.org/10.1038/ni.1856
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