Direct priming of antiviral cd8+ t cells in the peripheral interfollicular region of lymph nodes

ABSTRACT It is uncertain how antiviral lymphocytes are activated in draining lymph nodes, the site where adaptive immune responses are initiated. Here, using intravital microscopy we show

that after infection of mice with vaccinia virus (a large DNA virus) or vesicular stomatitis virus (a small RNA virus), virions drained to the lymph node and infected cells residing just

beneath the subcapsular sinus. Naive CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region and then formed tight interactions with dendritic cells, leading

to complete T cell activation. Thus, antigen presentation at the lymph node periphery, not at lymphocyte exit sites in deeper lymph node venules, as dogma dictates, has a dominant function

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Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INTERFERON-Γ COUPLES CD8+ T CELL AVIDITY AND DIFFERENTIATION DURING INFECTION Article Open access 23 October

2023 INNATE AND ADAPTIVE IMMUNE RESPONSES THAT CONTROL LYMPH-BORNE VIRUSES IN THE DRAINING LYMPH NODE Article Open access 25 June 2024 CD4+ T CELL CALIBRATION OF ANTIGEN-PRESENTING CELLS

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ACKNOWLEDGEMENTS We thank O. Schwartz, M. Czapiga, J. Kabat and S. Han for imaging advice and insight; D. Tokarchick and K. Irvine for technical assistance; and the staff of the Comparative

Medical Branch of the National Institute of Allergy and Infectious Diseases (building 33) for animal care. Supported by the Intramural Research Program of National Institute of Allergy and

Infectious Diseases. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, 20892, Maryland, USA Heather

D Hickman, Kazuyo Takeda, Cara N Skon, Faith R Murray, Scott E Hensley, Jack R Bennink & Jonathan W Yewdell * Department of Microbiology and Immunology and Sylvester Comprehensive

Cancer Center, University of Miami School of Medicine, Miami, 33136, Florida, USA Joshua Loomis & Glen N Barber Authors * Heather D Hickman View author publications You can also search

for this author inPubMed Google Scholar * Kazuyo Takeda View author publications You can also search for this author inPubMed Google Scholar * Cara N Skon View author publications You can

also search for this author inPubMed Google Scholar * Faith R Murray View author publications You can also search for this author inPubMed Google Scholar * Scott E Hensley View author

publications You can also search for this author inPubMed Google Scholar * Joshua Loomis View author publications You can also search for this author inPubMed Google Scholar * Glen N Barber

View author publications You can also search for this author inPubMed Google Scholar * Jack R Bennink View author publications You can also search for this author inPubMed Google Scholar *

Jonathan W Yewdell View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.D.H., J.W.Y. and J.R.B. conceptualized and designed the research;

H.D.H. did the IVM experiments and analyzed data; K.T. did the sectioning experiments (live and frozen); C.N.S. and F.R.M. prepared mice for IVM and aided in flow cytometry experiments;

S.E.H. did lymph flow experiments; J.L. and G.N.B. generated recombinant VSVs; and H.D.H., J.R.B. and J.W.Y. prepared the manuscript. CORRESPONDING AUTHOR Correspondence to Jonathan W

Yewdell. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–6 (PDF 998 kb) SUPPLEMENTARY VIDEO 1 Numerous slowly motile infected cells are found in the draining

inguinal lymph node after VV and VSV infection. Mice were either uninfected (left) or infected subcutaneously with 3.5 × 107 PFU of VV-NP-S-eGFP (middle) or 4 × 107 pfu of VSV-NP-S-eGFP

(right). The fusion protein produced by these viruses (consisting of the influenza A virus nucleoprotein (NP) in conjunction with the SIINFEKL peptide and eGFP) confers nuclear localization

to eGFP, resulting in the small, round appearance of eGFP+ cells. Also see the microscopic time course of infection (Fig. 1a,b) and cell speeds (Supplementary Fig. 1). (MOV 6810 kb)

SUPPLEMENTARY VIDEO 2 OT-I cells redistribute in the LN after VV infection. Mice were given 1.5 × 107 CMPTX-labeled OT-I cells 12 hours prior to infection with 3.5 × 107 PFU of VV-NP-S-eGFP

(middle) or 4.0 × 107 PFUof VSV-NP-S-eGFP (right). A serial scan from the lateral (outside) surface of the LN to the interior of the LN near the cortex shows the distribution of OT-I cells

(red) before (left) or approximately 6 hours post-infection. To identify the subcapsular sinus, these mice were also subcutaneously injected with wheat germ agglutinin (diffuse green

staining). Virus infected cells can be seen in the middle and right panels as distinct, bright green spots (corresponding to nuclei of infected cells). See also Figs. 2 and 4. (MOV 9303 kb)

SUPPLEMENTARY VIDEO 3 CD8+ T cells enter the node through HEVs located a greater depths beneath the SCS than the layer of VV-infected cells in the peri-SCS area. Mice were infected with

VV-NP-S-eGFP (green, nuclear) for 6 hours prior to the i.v. co-transfer of 1.5 × 107 OT-I cells (red) and FITC-dextran (green, vessel-contained). IVM images were obtained starting at 30

minutes post-transfer (time indicated in bottom right corner is time post-transfer). Left panel: 3-D projection of 0-150 μM is shown in the left panel, middle panel: projection of top 30 μm,

right panel: projection of 90-150 μm beneath the surface is shown in the right panel. Only OT-I cells present in vessels in the 90-150 μm sections of the node were seen leaving the HEV to

enter the ILN proper. Thus, IVM visualizes spatial separation between VV-infected cells, and deeper HEVs. See also Fig. 3. (MOV 6789 kb) SUPPLEMENTARY VIDEO 4 New CD8+ T cells are

continually recruited to the periphery of the node during infection. Mice were initially given 5 × 106 CMTPX-labeled OT-I cells (red). Twelve hours later, mice were infected with VV-ova (a

non-fluorescent VV expressing SIINFEKL). At 4 hours post-infection, mice were given 1.5 × 107 CMFDA-labeled OT-I cells (green, “late arrivers”). Newly transferred cells were allowed to

traffic within the animal/node for 4 hours prior to imaging. Within this time frame, many late-arriving CD8+ T cells were able to enter the node and traffic to the periphery to join in with

clusters of red OT-I (low resolution (20×) left; high resolution (63×) right). Even with 3-fold more green OT-I cells transferred after infection (late arrivers), the cells already present

in the node at the time of infection were far more prevalent in the periphery. (MOV 7226 kb) SUPPLEMENTARY VIDEO 5 Cognate antigen is required for CD8+ T cell redistribution and clustering

after infection. Mice were given 1.5 × 107 each of F5 (red) or OT-I cells (green). IVM images were acquired from 6-10 hours post-infection. 3-D projections are shown that were obtained from

uninfected mice (left panel), VV-ova-infected mice (middle panel), or NP(NT60)-infected mice (right panel). Note the accumulation of green OT-I cells when the ILN is infected with a VV

encoding SIINFEKL, and the likewise accumulation of red F5 cells when the VV encodes ASNENMDAM. See also Fig. 5. (MOV 8575 kb) SUPPLEMENTARY VIDEO 6 Antigen-specific CD8+ T cells move with

decreasing speeds with increasing proximity to the surface of the LN. Mice were given 1.5 × 107 CMPTX-labeled OT-I cells 12 hours prior to infection with 7 × 106 PFU of VV-NP-S-eGFP

(approximately 5-fold less than usual). At this virus dose, far fewer node cells are infected (shown in green). As a result, not all CD8+ T cells redistribute to the periphery of the LN, and

a large fraction remain deeper within the node. Shown are 5 μm sections taken at various depths beneath the SCS of the node (indicated at the top of the panel) over a 17 minute imaging

period. (MOV 6768 kb) SUPPLEMENTARY VIDEO 7 Cognate antigen is required for CD8+ T cell redistribution and clustering after VV infection. Mice were given 1.5 × 107 CMPTX-labeled OT-I cells

12 hours prior to infection with 3.5 × 107 PFU of VV-NP-S-eGFP or VV-eGFP. Images acquired 6-8 hours post-infection show that only when the cognate antigen for OT-I cells (SIINFEKL) is

present do they relocate to the periphery of the node, forming clusters with infected cells (compare left panel to center panel). Centrifugal redistribution also requires expression of the

restricting class I element, for VV-NP-S-eGFP infection results in very little redistribution to the periphery (and no stable contacts) in KbDb-KO mice. See also Supplementary Figs. 4 and 6.

(MOV 5026 kb) SUPPLEMENTARY VIDEO 8 Cognate antigen is required for CD8+ T cell redistribution and clustering after VSV infection. Mice were given 1.5 × 107 CMPTX-labeled OT-I cells 12

hours prior to infection with 4.0 × 107 PFU of VSV-NP-S-eGFP or VSV-eGFP. Images acquired 6-8 hours post-infection show that only when the cognate antigen for OT-I cells (SIINFEKL) is

present do they relocate to the periphery of the node, forming clusters with infected cells (compare left panel to center panel). Centrifugal redistribution also requires expression of the

restricting class I element, for VV-NP-S-eGFP infection results in very little redistribution to the periphery (and no stable contacts) in KbDb–KO mice. See also Supplementary Figs. 5 and 7.

(MOV 5037 kb) SUPPLEMENTARY VIDEO 9 Antigen-specific CD8+ T cells pursue rare, mobile virus-infected cells. Mice were given 1.5 × 107 CMPTX-labeled OT-I cells (red) 12 hours prior to

infection with 3.5 × 107 PFU of VV-NP-S-eGFP (green). Approximately 10 hours after infection, OT-I cells (red cells indicated by white circle) maintain contact with a motile virus-infected

cell moving in and out of the viewing area. See also Supplementary Figs. 5 and 7. (MOV 1563 kb) SUPPLEMENTARY VIDEO 10 Antigen-specific CD8+ T cells form long-lasting contacts with DCs after

VV-infection of the LN. CD11c-eGFP+ (shown, green DCs) or CD11c-eYFP+ mice were given 1.5 × 107 CMPTX-labeled OT-I cells (red) 12 hours prior to infection with 3.5 × 107 PFU of VV-ova

(expressing SIINFEKL, non-fluorescent). IVM images were acquired 12 h post-infection. Multiple OT-I cells cluster around 2 DCs in this field and maintain contact through the 46 minute

imaging period. See also Figs. 6 and 7. (MOV 4788 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hickman, H., Takeda, K., Skon, C. _et al._ Direct

priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes. _Nat Immunol_ 9, 155–165 (2008). https://doi.org/10.1038/ni1557 Download citation * Received: 18

September 2007 * Accepted: 11 December 2007 * Published: 13 January 2008 * Issue Date: February 2008 * DOI: https://doi.org/10.1038/ni1557 SHARE THIS ARTICLE Anyone you share the following

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