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Visualizing dendritic cell networks in vivo

Abstract

In the steady state, dendritic cells (DCs) in the lymph node induce T cell tolerance to self antigens. Innate signals trigger the maturation of tissue DCs, which migrate into lymph nodes and activate T cells. To examine DCs in vivo, we produced transgenic mice whose DCs expressed enhanced yellow fluorescent protein. Two-photon microscopy of lymph nodes in live mice showed that most of the steady-state DCs were enmeshed in an extensive network and remained in place while actively probing adjacent T cells with their processes. Mature DCs were more motile than steady-state DCs and were rapidly dispersed and integrated into the sessile network, facilitating their interaction with migrating T cells.

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Figure 1: Brightness and surface markers of CD11c-EYFP cells.
Figure 2: CD11c-EYFP mice examined by immunofluorescence.
Figure 3: Two-photon intravital microscopy of inguinal lymph nodes.
Figure 4: Anatomic distribution and activity of DCs in the lymph node.
Figure 5: DC clusters include more than one DC.
Figure 6: Transferred ECFP DCs gradually incorporate into the endogenous DC network.

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Acknowledgements

We thank R. Steinman for discussions and W. Gan for guidance on two-photon microscopy. Supported by the National Institutes of Health (AI55037 to M.L.D. and AI051573 to M.C.N.), Irene Diamond Foundation (M.L.D.), Rothschild Foundation (G.S.), Medical Scientist Training Program (GM07739 to R.L.L.), German Research Foundation (DU 548/1-1 to D.P.) and Howard Hughes Medical Institute (M.C.N.).

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Correspondence to Michael L Dustin or Michel C Nussenzweig.

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Supplementary information

Supplementary Fig. 1

Flow cytometric analysis of EYFP cells visible by two-photon microscopy. (PDF 366 kb)

Supplementary Fig. 2

Quantitative analysis of DC movement. (PDF 500 kb)

Supplementary Fig. 3

CD11c+ DCs form clusters with T and B cells in the steady state. (PDF 2360 kb)

Supplementary Video 1

Z-stack of two-photon images taken at depths of 0-300 μm through a B cell follicle in the LN of a living mouse. EYFP DCs are green, adoptively transferred EGFP B cells are false-colored cyan. Four populations of DCs are visible. Bright extended subcapsular DCs (arrows); dimmer DCs in a network surrounding the follicle; scattered compact DCs in the follicles (circles) and an extensive network of DCs in the T cell zone of the paracortex. (MOV 3861 kb)

Supplementary Video 2

Three-dimensional reconstruction of the different DC populations around a B cell follicle based on the data presented in Video 1. Colors are as in Video 1. (MOV 3453 kb)

Supplementary Video 3

No close association of resident CD11c-YFP cells with HEVs. Blood vessels were visualized with 66 kDa TRITC-dextran. The HEVs (crossing from top left to bottom right), can be distinguished from arterioles (crossing in the perpendicular direction) based on their irregular epithelium and the shadows of lymphocytes slowly rolling along it. YFP cellular debris is seen on the middle left. A mixture of slowly moving and sessile CD11c-YFP cells is seen scattered in the general area of the HEVs, but not immediately juxtaposed to them. Also seen are CFP lymphocytes (cyan) which exhibit a tighter morphology than DCs and can reach higher maximum speeds. (MOV 2956 kb)

Supplementary Video 4

Perifollicular DCs in relation to the LN capsule. A 3-dimensional reconstruction of a lymph node, showing the spatial relation of the perifollicular network of EYFP DCs (green) to the fibrous capsule (blue, second harmonics signal from collagen). (MOV 2480 kb)

Supplementary Video 5

Subcapsular sinus DCs and macrophages. Subcapsular sinus EYFP DCs (green) navigating among the less mobile macrophages, visualized by their phagocytosis of subcutaneously injected 66 kDa rhodamine-dextran (red). As in most videos below, a two-dimensional projection of a 50 μm thick volume is shown. (MOV 2793 kb)

Supplementary Video 6

Subcapsular DCs probing movement. At higher resolution, the probing movement of subcapsular DCs can be better appreciated. Note the characteristic ruffle shaped extensions of these cells. Dimmer, less motile perifollicular DCs are seen in the background. (MOV 3227 kb)

Supplementary Video 7

Tissue DC crawling. EYFP DCs (green) show fast crawling on the serosal surface lining the fat pads that neighbor the inguinal LN. Intravenously injected rhodamine dextran (red) flows in thin capillaries, bright red spots represent endocytosed dextran within endothelial cells. The morphology of these cells resembles that of subcapsular DCs in the LN. (MOV 1642 kb)

Supplementary Video 8

T cell zone DCs. EYFP DCs (green) form an extensive network in the T cell zone of the LN. Note that the DCs exhibit extensive probing movements but show little crawling. (MOV 3825 kb)

Supplementary Video 9

T cell zone DC network dynamics. A time sequence depicting a two-dimensional projection of a 50 μm volume in the interface of the T cell and B cell zones. The T cell zone is located below and to the left of the B cell follicle. The behavior of CD11c-EYFP DCs (green) in the network was followed. The great majority of the cells are laterally stable, exhibiting only probing movement, but occasionally a cell could be seen repositioning within the network (red circles). Adoptively transferred EGFP B cells are false-colored cyan. (MOV 2577 kb)

Supplementary Video 10

Different movement patterns of follicular and perifollicular DCs. Different behaviors of EYFP DCs (green) and ECFP B cells (cyan) in a B cell follicle (top right) and the perifollicular network (bottom left). Whereas some crawling movement is observed in the follicle, the DCs in the perifollicular network appear more dendritic and mainly probe with their processes. (MOV 1039 kb)

Supplementary Video 11

DC cluster Z stack. A Z-stack (45 μm deep, at 1 μm intervals) through several DC clusters in the T-B interface area. Each cluster is made up of numerous tightly apposed DCs enveloping lymphocytes. (MOV 2407 kb)

Supplementary Video 12

The behavior of DC clusters in the T-B interface zone (50 μm thick volume). Cluster position was stable for the duration and remained the same when the area was imaged again 4 hours later. DCs can be seen joining (blue circles) or leaving (red circles) the cluster. The shadows of lymphocytes can be seen drawn into the clusters enveloped in DC processes (yellow circles). (MOV 3422 kb)

Supplementary Video 13

Three-dimensional reconstruction of a DC cluster from the T-B interface area. The source data are confocal images that optically sectioned an immunofluorescently stained frozen section. EYFP is green, CD3 is red, and B220 is blue. Several DCs form a cluster that encloses numerous B and T cells. (MOV 2920 kb)

Supplementary Video 14

Resident LN DCs vs. transferred mature DCs of splenic origin. Transferred ECFP+ DCs (cyan), immunomagnetically purified from the spleen of a mouse treated with a FLT3-L-secreting tumor are seen here moving among resident CD11c-EYFP cells (yellow). The sequence was recorded at a depth of 180-230 μm 48 h after cell transfer. In this particular field, transferred cells crawled slightly faster than residents. Most of the movement consists of process probing, with cells occasionally flowing their soma and nucleus into one of the processes. To emphasize cell morphology, the CFP channel is shown alone on the right. (MOV 2619 kb)

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Lindquist, R., Shakhar, G., Dudziak, D. et al. Visualizing dendritic cell networks in vivo. Nat Immunol 5, 1243–1250 (2004). https://doi.org/10.1038/ni1139

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