Abstract
Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169+ macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169+ macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.
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Acknowledgements
We thank M. Kronenberg (La Jolla Institute for Allergy & Immunology) and C.-H. Wong (Scripps Laboratories) for GSL-1′; L. Van Kaer (Vanderbilt University School of Medicine) for CD1d-deficient mice; and A. Bendelac (University of Chicago) for DN32.D3 cells. Supported by Cancer Research UK (F.D.B., and C399/A2291 and C5255/A10339 to V.C.), the European Molecular Biology Organization Young Investigator Programme (F.D.B), the Royal Society (F.D.B.), The Wellcome Trust (084923 to V.C., G.B. and F.D.B.), the Ministerio de Education from Spain (2007-0148 to P.B.) and the European Commission (Seventh Framework Programme of the European Commission PIEF-GA-2008-220863 to P.B. and Immunanomap MRTN-CT-2006-035946 to P.P.).
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P.B. and F.D.B. design and conceived of the research in consultation with V.C. and P.P.; P.B. did all experiments; P.P. made initial observations that led to the study development and provided reagents; A.B. assisted with the multiphoton microscopy; N.v.R. provided clodronate liposomes; G.S.B. provided Gal(α1→2)α-GalCer; and P.B. and F.D.B. prepared the manuscript (in consultation with V.C. and P.P.).
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 (PDF 1005 kb)
Supplementary Movie 1
Dynamics of iNKT-S1 cells in resting LNs. iNKT-S1 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients and mediastinal LNs were imaged by multi-photon microscopy. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 3223 kb)
Supplementary Movie 2
Dynamics of iNKT-S2 cells in resting LNs. iNKT-S2 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients and mediastinal LNs were imaged by multi-photon microscopy. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 1662 kb)
Supplementary Movie 3
iNKT-S2 cells are arrested following stimulation with specific antigen. iNKT-S2 cells (red) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with α-GalCer (right panel). Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of iNKT movement are traced in pale red. Long ticks represent 20 μm. (MOV 1887 kb)
Supplementary Movie 4
iNKT-S1 cells are arrested following stimulation with specific antigen. iNKT-S1 cells (red) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with α-GalCer. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of iNKT movement are traced in pale red. Long ticks represent 20 μm. (MOV 734 kb)
Supplementary Movie 5
iNKT-S2 cells arrest in response to specific antigen is dependent on CD1d expression. iNKT-S2 cells (red) and CD4+ T cells (blue) were adoptively transferred into CD1d-KO recipients, prior to i.p. injection of particles coated with α-GalCer. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 1777 kb)
Supplementary Movie 6
iNKT-S2 cells are retained at the SCS in response to specific antigen. iNKT-S2 cells (red) and B cells (blue) were adoptively transferred into WT recipients, prior to injection of particles containing α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 2 h after particle administration. Two different examples are shown (left panel and right panels) with XY (upper panels) and XZ (bottom panels) projections of the imaged volumes. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 5701 kb)
Supplementary Movie 7
iNKT-S2 cells exhibit long-lasting arrests at the SCS in response to specific antigen. iNKT-S2 cells (red) and B cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 16 h after particle administration. A movie of the XY projection (left) is shown together with three-dimensional representations of the imaged volume (right and middle). Representative tracks of cell movement corresponding to the full length of the movie are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 11568 kb)
Supplementary Movie 8
iNKT-S1 cells exhibit long-lasting arrests at the SCS in response to specific antigen. iNKT-S1 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 16 h after particle administration. A movie of the XY projection (left) is shown together with a three-dimensional representation (right) of the imaged volume. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 5060 kb)
Supplementary Movie 9
iNKT cells exhibit long-lasting contacts with CD169+ SCS macrophages in response to specific antigen administration. iNKT-S2 cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with αGalCer (red). Animals received anti-mouse CD169 antibody (green) 15 min before imaging. Draining LNs were imaged by multi-photon microscopy 6 h after particle administration. Movies with XY (left panels) and XZ (right lower panel) projections are shown together with a three-dimensional representation of the imaged volume (right upper panel). Representative tracks of cell movement are traced. Long ticks represent 20 μm. (MOV 2874 kb)
Supplementary Movie 10
iNKT cells are retained at CD169+ SCS macrophages in response to specific antigen. iNKT-S2 cells (blue) were adoptively transferred into WT recipients, prior to injection of particles containing αGalCer (red). Animals received anti-mouse CD169 antibody (green) 15 min before imaging. Draining LNs were imaged by multi-photon microscopy 6 h after injection with particles. Long ticks represent 20 μm. (MOV 9467 kb)
Supplementary Movie 11
iNKT-S1 cells are arrested following stimulation with particulate GSL-1'. iNKT-S1 cells (red) together with CD4+ T cells (blue) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with GSL-1'. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. (MOV 1784 kb)
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Barral, P., Polzella, P., Bruckbauer, A. et al. CD169+ macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes. Nat Immunol 11, 303–312 (2010). https://doi.org/10.1038/ni.1853
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DOI: https://doi.org/10.1038/ni.1853
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