Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

doi:10.1128/JVI.01108-16

. 2016 Sep 29;90(20):9046-57.

doi: 10.1128/JVI.01108-16. Print 2016 Oct 15.

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

Clara Lawler¹, Cindy S E Tan¹, J Pedro Simas², Philip G Stevenson³

Affiliations

¹ School of Chemistry and Molecular Biosciences, University of Queensland and Royal Children's Hospital, Brisbane, Australia.
² Instituto de Medicina Molecular e Instituto de Microbiologia, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
³ School of Chemistry and Molecular Biosciences, University of Queensland and Royal Children's Hospital, Brisbane, Australia p.stevenson@uq.edu.au.

PMID: 27466430
PMCID: PMC5044861
DOI: 10.1128/JVI.01108-16

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

Clara Lawler et al. J Virol. 2016.

. 2016 Sep 29;90(20):9046-57.

doi: 10.1128/JVI.01108-16. Print 2016 Oct 15.

Authors

Clara Lawler¹, Cindy S E Tan¹, J Pedro Simas², Philip G Stevenson³

Affiliations

¹ School of Chemistry and Molecular Biosciences, University of Queensland and Royal Children's Hospital, Brisbane, Australia.
² Instituto de Medicina Molecular e Instituto de Microbiologia, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
³ School of Chemistry and Molecular Biosciences, University of Queensland and Royal Children's Hospital, Brisbane, Australia p.stevenson@uq.edu.au.

PMID: 27466430
PMCID: PMC5044861
DOI: 10.1128/JVI.01108-16

Abstract

Gammaherpesviruses establish persistent, systemic infections and cause cancers. Murid herpesvirus 4 (MuHV-4) provides a unique window into the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defense. Type I interferon (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required, but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when both IFN-I responses and NK cells were lacking. Thus, multiple innate defenses allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and thus improve infection control.

Importance: Human gammaherpesviruses cause cancers by infecting B cells. However, vaccines designed to block virus binding to B cells have not stopped infection. Using a related gammaherpesvirus of mice, we have shown that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gammaherpesvirus production by recruiting type I interferons and natural killer cells. Therefore, a vaccine that speeds the recruitment of these defenses might stop B cell infection.

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Figures

**FIG 1**
IFNAR blockade increases PLN and spleen infections by i.f. MuHV-4. (a) C57BL/6 mice given IFNAR-blocking antibody (αIFN) or not (cont) were infected i.f. with MuHV-4 (10⁵ PFU). Six days later, PLN and spleen viruses were IC assayed for total virus and plaque assayed for infectious virus. Crosses show means, and other symbols show data for individual mice. IFNAR-blocking antibody increased total but not lytic titers. (b) Mice treated as described for panel a were plaque assayed 3 and 6 days later for infectious virus in footpads. Bars show means, and diamonds show data for individuals. IFNAR-blocking antibody increased titers at both time points. (c) Mice treated as described for panel a were IC assayed for reactivatable virus in PLN and spleen 3 and 6 days later. Bars show means; other symbols show data for individuals. IFNAR-blocking antibody increased infection at both time points in both sites. (d) Mice treated as described for panel a were tested 1 day later for infectious footpad virus by plaque assay and for reactivatable PLN virus by IC assay. Crosses show means; other symbols show data for individual mice. IFNAR-blocking antibody increased PLN but not footpad infection. (e) DNA from PLN in panel d was assayed for viral genome load by quantitative PCR. Viral load (K3) was normalized by cellular load (β-actin) for each sample. Crosses show means; other symbols show data for individuals. IFNAR-blocking antibody increased viral genome loads. (f) Mice were given IFNAR-blocking antibody or not and then latency-deficient MHV-M50 (10⁵ PFU i.f.). One day later, footpad virus was plaque assayed and PLN virus was IC assayed. Crosses show means; other symbols show data for individuals. IFNAR-blocking antibody increased PLN but not footpad infection. (g) Mice were depleted of pDC (αpDC) or not (cont) by 2 i.p. injections of anti-CD317/BST-2 MAb (400 μg/mouse) 48 h apart and then given MHV-GFP i.f. (10⁵ PFU). Three days later, footpad virus was plaque assayed, and PLN virus and spleen virus were IC assayed. Bars show means; other symbols show data for individual mice. pDC depletion increased footpad but not PLN or spleen infections. Two further experiments gave equivalent results. (h) pDC depletion efficacy was checked by flow cytometry of gated CD11c⁺ spleen cells for the pDC markers Siglec-H and BST-2. n is the number of cells in the boxed region.

**FIG 2**
IFNAR blockade increases SSM infection. (a) Mice given IFNAR-blocking antibody (αIFN) or not (cont) were infected i.f. with MHV-GFP (10⁵ PFU). One day later, PLN sections were stained for viral GFP. Nuclei were stained with DAPI. Gray arrows show example GFP⁺ cells in the LN substance. Open arrows show increased GFP staining around the subcapsular sinus of mice treated with IFNAR-blocking antibody. Each image is representative of data for 6 samples per group. (b) GFP⁺ cells were counted for samples as described for panel a. Bars show group means. Other symbols show mean counts for 3 randomly selected fields of view per section across 3 sections of individual mice. IFNAR-blocking antibody significantly increased GFP⁺ cell numbers. (c) C57BL/6 mice were given IFNAR-blocking antibody or not and infected or not with MuHV-4 as described for panel a. Six days later, PLN sections were stained for CD169 (SSM) and CD68 (macrophages/DC). Infected mice lost CD169 expression around the subcapsular sinus (arrows), regardless of IFNAR blockade. CD68 staining around the subcapsular sinus was also reduced. (d) PLN from mice infected as described for panel a were stained 1 day later for GFP plus CD68, CD169, or B220 (B cells). Arrows show example dual-positive cells. (e) GFP⁺ and viral lytic antigen (MHV)-positive cells colocalizing with myeloid cell markers were counted for 3 fields of view per section across 3 sections for each of 5 mice per group. Bars show group means; other symbols show individual mean counts. IFNAR-blocking antibody increased GFP⁺ and MHV⁺ myeloid (CD169⁺ or CD68⁺) cell numbers around the subcapsular sinus. (f) PLN of mice infected as described for panel a were stained 1 day later for viral GFP and lytic antigens (MHV). MHV expression was minimal in control mice. Arrows show example GFP⁺ MHV⁺ cells in mice treated with IFNAR-blocking antibody. Each image is representative of data for 6 samples per group. (g) PLN of mice infected as described for panel a were stained for viral antigens and cell type markers. Arrows show examples of colocalization. Quantitation was as described for panel e.

**FIG 3**
IFN-I restricts SSM infection independently of viral lytic gene expression. (a) Mice were given IFNAR-blocking antibody (αIFN) or not (cont) and then given i.f. ORF50⁻ MuHV-4 (10⁵ PFU), which expresses lytic genes only with complementation. One day later, PLN sections were stained for viral GFP. Arrows show example GFP⁺ cells. Each image is representative of results for 6 samples per group. (b) GFP⁺ cells were counted across 3 randomly selected fields of view per section for 3 sections from each of 6 mice per group. Bars show group means, and other symbols show mean counts for individuals. IFNAR-blocking antibody increased GFP⁺ cell numbers. (c) PLN of mice infected as described for panel a were stained for viral GFP plus CD68, CD169, and B220. Arrows show example dual-positive cells. No B cells were GFP⁺.

**FIG 4**
IFNAR blockade and SSM depletion synergistically disseminate MuHV-4. (a) Mice were given liposomal clodronate to deplete SSM (clod), IFNAR-blocking antibody (αIFN), both treatments (both), or neither treatment (cont) and then given MHV-GFP i.f. (10⁵ PFU). Six days later, footpad virus was plaque assayed, and PLN and spleen virus were IC assayed. Bars show means, and other symbols show individual titers. All treatments increased footpad and spleen titers; only dual treatment increased PLN titers. *, P < 0.05; **, P < 0.01; ***, P < 10⁻³; ****, P < 10⁻⁴. (b) GFP⁺ and viral antigen (MHV)-positive cells were counted on PLN sections of mice treated as described for panel a. Bars show group means. Other symbols show mean counts for 3 fields of view per section across 3 sections of each mouse. All treatments increased GFP⁺ and MHV⁺ cell numbers, although MHV⁺ cell numbers were low. (c) Example images from panel b show infected cells in PLN. Most GFP⁺ cells were B220⁺ (B cells, white arrows). Gray-filled arrows show lytically infected cells. (d) GFP⁺ and MHV⁺ cells were counted on spleen sections of mice treated as described for panel a. Bars show group means. Other symbols show mean counts for 3 fields of view per section across 3 sections of individual mice. Only dual treatment (both) increased spleen infection by this measure. (e) Splenic GFP⁺ cells were further subdivided by site. Dual treatment increased infection in the red pulp (RP), MZ, and white pulp (WP). Single treatments had no significant effect. (f) Example images from panel e show infected B220⁺ cells. Dashed lines correspond to the MZ. White arrows show infected WP B cells. The gray-filled arrow shows a lytically infected cell.

**FIG 5**
IFNAR blockade increases virus production in but not transfer from LysM⁺ cells. (a) LysM-cre mice were given IFNAR-blocking antibody (αIFN) or not (cont) and then floxed color-switching MHV-RG (10⁵ PFU i.f.). Six days later, virus was plaque assayed (footpads) or IC assayed (PLN and spleens). Horizontal bars show means. Other symbols show data for individual mice. The dashed line indicates the assay sensitivity limit. IFNAR-blocking antibody increased infection in footpads and spleens but not PLN. (b) Viruses from panel a were assayed for fluorochrome switching. Bars show means; other symbols show data for individual mice. IFNAR-blocking antibody increased virus switching in footpads and PLN. ND, not determined, as there was insufficient virus. (c) Tissue sections of mice infected as described for panel a were analyzed for infected-cell fluorochrome expression. Bars show group means. Other points show means of data for 3 views per section for each of 3 sections per mouse. IFNAR-blocking antibody increased fluorochrome switching in both PLN and spleens. (d) Example PLN images show unswitched (mCherry⁺, white arrows) but not switched (GFP⁺) CD11c⁺ cells in control mice and mice treated with IFNAR-blocking antibody. Gray arrows show example GFP⁺ cells.

**FIG 6**
IFNAR blockade increases virus production in and transfer from CD11c⁺ cells. (a) CD11c-cre mice were given IFNAR-blocking antibody or not and then MHV-RG i.f. (10⁵ PFU). Six days later, virus was plaque assayed (footpads) or IC assayed (PLN and spleens). Horizontal bars show means. Other symbols show data for individual mice. The dashed line indicates the assay sensitivity limit. IFNAR-blocking antibody significantly increased footpad and PLN but not spleen infections. (b) Viruses from panel a were assayed for fluorochrome switching. Bars show means; other symbols show data for individuals. IFNAR-blocking antibody significantly increased the switching of virus recovered from spleens. (c) Tissue sections of mice infected as described for panel a were analyzed for cellular fluorochrome expression. Bars show group means. Other points show mean counts for 3 views per section for 3 sections per mouse. IFNAR-blocking antibody increased infected-cell switching in both PLN and spleens. (d) Spleens were analyzed further for viral fluorochrome-positive cell types. IFNAR-blocking antibody increased B220⁺ and CD11c⁺ cell switching, although the increase was significant only for CD11c⁺. (e) PLN overview images show more GFP⁺ (switched) cells with IFNAR-blocking antibody, quantitated as described for panel c. Unswitched (mCherry⁺) cell numbers were similar to those of controls. Six mice per group gave equivalent results. (f) Higher-power images show both switched and unswitched B cells with IFNAR-blocking antibody and only unswitched B cells in controls. The images are representative of results for 6 mice per group. (g) Spleen overview images show IFNAR-blocking antibody increasing switched GFP⁺ cell numbers in the MZ between WP follicles and the F4/80⁺ red pulp (arrows). Six mice per group gave similar results, quantitated as described for panel c. (h) Higher-power spleen images show example GFP⁺ and mCherry⁺ cells. GFP⁺ cells were evident in all mice, but IFNAR-blocking antibody gave significantly more CD11c⁺ GFP⁺ cells than controls, quantitated as described for panel d.

**FIG 7**
IFN-I and NK cells control PLN infection. (a) C57BL/6 mice were given IFNAR-blocking antibody (αIFN) or NK-depleting antibody (αNK) or left untreated (cont) and then infected i.f. with MHV-GFP (10⁵ PFU). One day later, PLN sections were stained for viral GFP plus NKp46⁺ NK cells (NK), myeloid cells (CD68), or SSM (CD169). Nuclei were stained with DAPI. Open arrows show NKp46⁺ NK cells around the subcapsular sinus. Gray-filled arrows show CD68⁺ and CD169⁺ infected cells. (b) NKp46⁺ NK cells were counted on PLN sections of mice treated as described for panel a. Bars show group means. Circles show mean counts for 3 fields of view per section from 3 sections per mouse. IFNAR-blocking antibody significantly increased NK cell recruitment above controls, whereas NK depletion significantly reduced it. (c) Spleen cells of mice given anti-NK1.1 depleting antibody (PK136) (200 μg/mouse in 2 injections 48 h apart) or not (control) were analyzed 24 h later by flow cytometry for expression of the NK cell marker NKp46. n is the number of cells in the boxed region. (d) PLN of mice treated as described for panel a were IC assayed for recoverable virus 1 day after i.f. MHV-GFP. Crosses show means; other symbols show data for individuals. Both NK cell-depleting antibody and IFNAR-blocking antibody increased titers. IFNAR-blocking antibody had a significantly greater effect. (e) GFP⁺ cells on PLN sections described for panel a were counted for 3 views per section across 3 sections per mouse. Circles show mean counts for individuals. Bars show group means. Both IFNAR-blocking antibody and NK cell-depleting antibody increased GFP⁺ cell numbers. IFNAR-blocking antibody had a significantly greater effect.

**FIG 8**
SSM pass on infection when both IFN-I and NK cells are disabled. (a) LysM-cre mice were given IFNAR-blocking (αIFN) and NK cell-depleting (αNK) antibodies or not and then infected i.f. with MHV-RG (10⁵ PFU). Four days later, infectious virus from footpads was plaque assayed, and reactivatable virus from PLN and spleen virus were IC assayed. Crosses show means; other symbols show data for individuals. IFNAR-blocking and NK cell-depleting antibodies significantly increased PLN and spleen titers. The baseline is the assay sensitivity limit. (b) Virus described for panel a was typed for fluorochrome expression. IFNAR-blocking and NK cell-depleting antibodies increased fluorochrome switching in PLN, and in mice treated with IFNAR-blocking and NK cell-depleting antibodies, spleen virus was significantly more switched than PLN virus. Control mice yielded insufficient spleen virus for assays (ND, not determined). (c) PLN sections of mice treated as described for panel a were stained for virus-expressed GFP (switched) and mCherry (unswitched). Total GFP⁺ cells were counted for 3 fields of view per PLN section across 3 sections for each of 6 mice (left-hand graph). Bars show mean counts ± standard errors of the means for individual mice. IFNAR-blocking and NK cell-depleting antibodies significantly increased total GFP⁺ cell numbers. However, the ratios of GFP⁺ cells to mCherry⁺ cells (percentages of switched cells) (right-hand graph) were not significantly different. Circles and squares show mean counts for individuals, and bars show group means. (d) PLN sections of mice treated as described for panel a were stained for GFP and CD169 to identify fluorochrome-switched, infected SSM. Arrows show examples. Total GFP expression was quantitated as described for panel c. (e) PLN sections of mice treated as described for panel a were stained for GFP and B220 to identify fluorochrome-switched, infected B cells. Symbols show mean counts for 2 fields of view per mouse. Bars show group means. Control mice lacked GFP⁺ B cells. (f) The arrow shows an example GFP⁺ B220⁺ cell in PLN of an IFNAR-blocked, NK cell-depleted mouse, quantitated as described for panel d.

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References

1. Cesarman E, Mesri EA. 2007. Kaposi sarcoma-associated herpesvirus and other viruses in human lymphomagenesis. Curr Top Microbiol Immunol 312:263–287. - PubMed
1. Joseph AM, Babcock GJ, Thorley-Lawson DA. 2000. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils. J Virol 74:9964–9971. doi:10.1128/JVI.74.21.9964-9971.2000. - DOI - PMC - PubMed
1. Balfour HH. 2014. Progress, prospects, and problems in Epstein-Barr virus vaccine development. Curr Opin Virol 6:1–5. doi:10.1016/j.coviro.2014.02.005. - DOI - PMC - PubMed
1. Dunmire SK, Grimm JM, Schmeling DO, Balfour HH Jr, Hogquist KA. 2015. The incubation period of primary Epstein-Barr virus infection: viral dynamics and immunologic events. PLoS Pathog 11:e1005286. doi:10.1371/journal.ppat.1005286. - DOI - PMC - PubMed
1. McGeoch DJ. 2001. Molecular evolution of the gamma-Herpesvirinae. Philos Trans R Soc Lond B Biol Sci 356:421–435. doi:10.1098/rstb.2000.0775. - DOI - PMC - PubMed

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[1] Cesarman E, Mesri EA. 2007. Kaposi sarcoma-associated herpesvirus and other viruses in human lymphomagenesis. Curr Top Microbiol Immunol 312:263–287. - PubMed

[2] Cesarman E, Mesri EA. 2007. Kaposi sarcoma-associated herpesvirus and other viruses in human lymphomagenesis. Curr Top Microbiol Immunol 312:263–287. - PubMed

[3] Joseph AM, Babcock GJ, Thorley-Lawson DA. 2000. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils. J Virol 74:9964–9971. doi:10.1128/JVI.74.21.9964-9971.2000. - DOI - PMC - PubMed

[4] Joseph AM, Babcock GJ, Thorley-Lawson DA. 2000. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils. J Virol 74:9964–9971. doi:10.1128/JVI.74.21.9964-9971.2000. - DOI - PMC - PubMed

[5] Balfour HH. 2014. Progress, prospects, and problems in Epstein-Barr virus vaccine development. Curr Opin Virol 6:1–5. doi:10.1016/j.coviro.2014.02.005. - DOI - PMC - PubMed

[6] Balfour HH. 2014. Progress, prospects, and problems in Epstein-Barr virus vaccine development. Curr Opin Virol 6:1–5. doi:10.1016/j.coviro.2014.02.005. - DOI - PMC - PubMed

[7] Dunmire SK, Grimm JM, Schmeling DO, Balfour HH Jr, Hogquist KA. 2015. The incubation period of primary Epstein-Barr virus infection: viral dynamics and immunologic events. PLoS Pathog 11:e1005286. doi:10.1371/journal.ppat.1005286. - DOI - PMC - PubMed

[8] Dunmire SK, Grimm JM, Schmeling DO, Balfour HH Jr, Hogquist KA. 2015. The incubation period of primary Epstein-Barr virus infection: viral dynamics and immunologic events. PLoS Pathog 11:e1005286. doi:10.1371/journal.ppat.1005286. - DOI - PMC - PubMed

[9] McGeoch DJ. 2001. Molecular evolution of the gamma-Herpesvirinae. Philos Trans R Soc Lond B Biol Sci 356:421–435. doi:10.1098/rstb.2000.0775. - DOI - PMC - PubMed

[10] McGeoch DJ. 2001. Molecular evolution of the gamma-Herpesvirinae. Philos Trans R Soc Lond B Biol Sci 356:421–435. doi:10.1098/rstb.2000.0775. - DOI - PMC - PubMed

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Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

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Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

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