Despite intense interest in antiviral T cell priming, the routes by which virions move in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined the locations of virions and infected cells after administration to mice of vaccinia virus or Zika virus. Notably, many rapidly infected cells in the LN interior were adjacent to LN conduits. Through the use of confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia virus–infected cells in the LN paracortex, which led to T cell activation in the LN interior. These results reveal that it is possible for even large virions to flow through LN conduits and infect dendritic cells within the T cell zone to prime CD8+ T cells.
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The data that support the findings of this study are available from the corresponding authors upon reasonable request.
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This work was supported by the Intramural Research Program of NIAID, NIH (H.D.H.). R.V.S. was supported by NIH grants R21CA172983, R21CA175592, R01GM120592, and AHA 16GRNT27260362. We thank K. Dowd and T. Pierson (Viral Pathogenesis Section, Laboratory of Viral Diseases, NIAID) for assistance with ZIKV infection and J. Kabat (Biological Imaging Section, NIAID) for assistance with microscopy image analysis. G. Oliver (University of Chicago) provided Prox1-CreERT2tg/+ mice on the C57BL/6 genetic background.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Staining with an antibody to VACV-Ag confirms infection of cells expressing viral promoter–driven eGFP.
a) Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 8 h after footpad infection with VACV-NP-S-eGFP. ERTR7 = red, rabbit polyclonal antibody against total VACV antigens = white, VACV-infected cells = green nuclear signal, B220 = blue. Scalebar = 200 μm. Boxes highlight different areas of the node in which infected cells are located. Higher magnification images are shown in the insets. Scalebars = 20 μm. For ease of viewing VACV-infected cells, the VACV-antigen-antibody signal (white) is omitted on the same inset image on the right hand side. b) As in a) but larger deconvolved images showing nuclear eGFP signal surround by white, cytoplasmic VACV Ag staining (VACV replicates in the cytoplasm). VACV eGFP signal (green) is omitted on the right. Scalebars = 20 μm. Images are representative of 6 nodes from 3 different experiments.
Supplementary Figure 2 VACV-infected cells can be visualized in all classified nodal locations by 8 h after infection.
Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 8 h after footpad infection with VACV-NP-S-eGFP. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green nuclear signal, B220 = blue. Scalebar = 200 μm. Boxes highlight different areas of the node in which infected cells are located. Higher magnification images are shown corresponding to boxed areas. Scalebars = 20 μm. Left panels show merged image; right panels show only B cells + conduits + VACV-infected cells. Images are representative of 6 nodes from 3 different experiments.
Supplementary Figure 3 GFP+ virus-infected cells can be visualized in the T cell zone by 4 h after infection.
Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 4 h after footpad injection of MVA-(a-b) or VACV (c-d). ERTR7 = red, Lyve-1 = white, VACV- or MVA-infected cells = green nuclear signal, B220 = blue. Arrows point to some of the T cell zone virus-infected cells associated with conduits. Scalebars = 100 μm. Higher magnification images are shown to the right in (b and d). Scalebars = 15 μm. e) Deconvolved, high-magnification, blended-view images showing the association of T cell zone MVA-infected cells with conduits at 4 h p.i. Scalebar = 20 μm. Boxes indicate areas with increased magnification on the right. Scalebars = 5 μm. Images are representative of 6 nodes from 3 different experiments.
a-b) Transmission electron micrographs of sections of popliteal LNs harvested 5 m after footpad infection of 108 PFU of 10 nM gold particle-conjugated VACV virions (indicated with an arrow). Panel a) shows a magnified image of panel b). Panel a) Scalebars = 200 nm. b) = 1 μm. Arrows indicate the same VACV virion in the two images. The conduits (labeled with a C) can be identified by organized collagen structures running either parallel (a-d) or anti-parallel (e-f) to the field of view and are lined by fibroblastic reticular cells (FRC). T = T cell. c-d) Transmission electron micrograph as in panel a-b, but in a different area of the T cell zone. Panel c) shows a magnified image of panel d). Panel c) Scalebars = 100 nm. d) = 1 μm. An arrow points to the same VACV virion in the two images. e-f) Transmission electron micrographs showing that cells near the conduits (labeled C) in the LN T cell zone of the node acquire VACV (indicated by arrows). In these images, the collagen in the conduit is running anti-parallel to the images and the FRCs that ensheath the conduit can be seen. Panel e) Scalebars = 500 nm. f) = 2 μm. Results are indicative of 14 grids from 2 separate experiments.
Supplementary Figure 5 Single-color stains for cell-surface markers on VACV-infected cells in the LN.
a-f) Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 8 h after footpad injection of 108 PFU of VACV. Different cell surface markers are shown in white (indicated above each image). Staining in white for a) CD11b, b) CD169, c) F4/80, d) CD11c, e) CD205, f) SIGNR1. Scalebars = 100 μm. VACV-infected cells = green, B220 = blue. Some of the infected cells positive for each surface marker are indicated by tan circles. Higher magnification images of infected cells are shown below the whole LN section. Scalebars = 20 μm. Images are representative of 10 nodes from 4 different experiments.
a) Maximum intensity projection (MIPs) of a section of a popliteal LN of an Ifnar -/- mouse infected with 104 FFU ZIKV in the footpad. LNs were harvested 24 h post-infection. Section was stained with secondary Ab only to show background staining for ZIKV-infected cells (white). Insets show higher magnification view without (left) or with (right) staining for CD11c (green). ERTR7 = red, B220 = blue. Scalebars = 200 μm (left), 20 μm (right higher mag.) Image is representative of 3 nodes from 2 different experiments. b-e) Electron micrographs of LNs infected and harvested as in (a). Conduits are labeled with a “C.” Arrows point to virions in the conduit. For clarity, the borders of conduits are lined with yellow dashed lines. b) Middle and right panels show higher magnification views of the same conduit in the left panels. ZIKV virions can be seen at three locations in this conduit. Left panel scalebar = 2 μm, middle = 500 nm, middle inset = 100 nm, right = 500 nm, right inset =100 nm. c) Right panel (scalebar= 500 nm) shows higher magnification view of left panel (scalebar = 100 nm). d-e) Two different conduits with ZIKV virions. Left scalebar = 200 nm, right scalebar = 50 nm. 24 grids from 6 nodes and 3 experiments were analyzed.
Supplementary Figure 7 CD69 expression is highest on T cells in the T cell zone at 8 h after infection.
a) Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 8 h after footpad injection of 108 PFU of VACV-NP-S-eGFP. OT-I CD8+ T cells = red, VACV-infected cells = green, CD69 = white, B220 = blue. Left panel) all stains merged. Right panel) OT-I CD8+ T cell activation was assessed by colocalization of the red signal with the white signal (with the colocalized signal shown in purple). Arrows point to some of the activated CD8+ T cells. Scalebars = 100 μm. 10 LNs from 5 experiments were analyzed. b) MIP as in (a) with a VACV lacking SIINFEKL (VACV-NP-eGFP). Color-coded numbers indicating the CD69 mean fluorescent intensity (MFI) for each T cell (with SCS&IFA numbers in pink, medulla in green, T cell zone in yellow, B cell follicles in blue). Scalebar = 200 μm. c) Quantification of the CD69 MFIs of all OT-I CD8+ T cells in either the SCS&IFA (red), medulla (green), T cell zone (yellow), or B cell follicle (blue). Dots = Individual T cells from 1 LN section. n = 195 total T cells. Bar = mean. Statistics were calculated using a Mann-Whitney test. 6 LNs from 3 experiments were analyzed. d-e) as in (b-c) except with a VACV expressing SIINFEKL (VACV-NP-S-eGFP). Scalebar = 200 μm. e) Dots = Individual T cells from 1 LN section. n = 187 total T cells. Bar = mean. Statistics were calculated using a Mann-Whitney test. 6 LNs from 3 experiments were analyzed.
Supplementary Figure 8 OT-I CD8+ T cells are activated mainly in the LN SCS and IFA region at a low dose of virus and in the T cell zone at a high dose.
a) Maximum intensity projections (MIPs) of sections of popliteal LNs harvested 8 h after footpad injection of the indicated dose of VACV (top panels) or MVA (bottom panels). ERTR7 = white, infected cells = green nuclear signal. Left panels at each viral dose = merge; right panels = only infected cells. Scalebars = 100 μm. b) Number of infected cells at each LN location with varying viral dose. Dots represent the number of infected cells per LN section when popliteal LNs were harvested at 8 h post-footpad infection. n = 10 LNs/dose for all infections except: VACV at 108 PFU (n = 18); MVA and VACV at 107 PFU (n = 12). LNs came from at least 2 different experiments per group. c) The total number of infected cells per LN section correlates with the number of infected cells located in the T cell zone. Data from all viral doses in (b) were combined to create (c). d) MIPs of sections of popliteal LNs harvested 8 h after footpad injection at the indicated dose of VACV. OT-I cells = red, VACV-infected cells = green, B220 = blue, CD69 = white. Scalebars = 100 μm. The boxed area of the LN is magnified in the images below each section. Scalebars = 20 μm. In the far-right panels, OT-I CD8+ T cell activation was assessed by colocalization of the red signal with the white signal (with the colocalized signal shown in purple). Arrows point to activated CD8+ T cells. (Note that VACV-NP-eGFP does not contain SIINFEKL and should not activate OT-I CD8+ T cells, but does activate endogenous VACV-specific CD8+ T cells). Images are representative of 5 LNs per group from 2 different experiments.