Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul;88(14):8016-27.
doi: 10.1128/JVI.00913-14. Epub 2014 May 7.

Role of CD8+ T cells and lymphoid dendritic cells in protection from ocular herpes simplex virus 1 challenge in immunized mice

Harry Matundan  1 , Kevin R Mott  1 , Homayon Ghiasi  2
Affiliations

Role of CD8+ T cells and lymphoid dendritic cells in protection from ocular herpes simplex virus 1 challenge in immunized mice

Harry Matundan et al. J Virol. 2014 Jul.

Abstract

The development of immunization strategies to protect against ocular infection with herpes simplex virus 1 (HSV-1) must address the issue of the effects of the strategy on the establishment of latency in the trigeminal ganglia (TG). It is the reactivation of this latent virus that can cause recurrent disease and corneal scarring. CD8(+) T cells and dendritic cells (DCs) have been implicated in the establishment and maintenance of latency through several lines of inquiry. The objective of the current study was to use CD8α(-/-) and CD8β(-/-) mice to further evaluate the contributions of CD8(+) T cells and the CD8α(+) and CD8α(-) subpopulations of DCs to the protection afforded against ocular infection by immunization against HSV-1 and their potential to increase latency. Neutralizing antibody titers were similar in immunized CD8α(-/-), CD8β(-/-), and wild-type (WT) mice, as was virus replication in the eye. However, on day 3 postinfection (p.i.), the copy number of HSV-1 glycoprotein B (gB) was higher in the corneas and TG of CD8α(-/-) mice than those of WT mice, whereas on day 5 p.i. it was lower. As would be anticipated, the lack of CD8α(+) or CD8β(+) cells affected the levels of type I and type II interferon transcripts, but the effects were markedly time dependent and tissue specific. The levels of latent virus in the TG, as estimated by measurement of LAT transcripts and in vitro explant reactivation assays, were lower in the immunized, ocularly challenged CD8α(-/-) and WT mice than in their CD8β(-/-) counterparts. Immunization reduced the expression of PD-1, a marker of T-cell exhaustion, in the TG of ocularly challenged mice, and mock-immunized CD8α(-/-) mice had lower levels of PD-1 expression and latency than mock-immunized WT or CD8β(-/-) mice. The expansion of the CD8α(-) subpopulation of DCs through injection of WT mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA reduced the amount of latency and PD-1 expression in the TG of infected mice. In contrast, injection of FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which expanded both subpopulations, was less effective. Our results suggest that the absence of both CD8α(+) T cells and CD8α(+) DCs does not reduce vaccine efficacy, either directly or indirectly, in challenged mice and that administration of GM-CSF appears to play a beneficial role in reducing latency and T-cell exhaustion. Importance: In the past 2 decades, two large clinical HSV vaccine trials were performed, but both vaccine studies failed to reach their goals. Thus, as an alternative to conventional vaccine studies, we have used a different strategy to manipulate the host immune responses in an effort to induce greater protection against HSV infection. In lieu of the pleiotropic effect of CD8α(+) DCs in HSV-1 latency, in this report, we show that the absence of CD8α(+) T cells and CD8α(+) DCs has no adverse effect on vaccine efficacy. In line with our hypothesis, we found that pushing DC subpopulations from CD8α(+) DCs toward CD8α(-) DCs by injection of GM-CSF reduced the amount of latent virus and T-cell exhaustion in TG. While these studies point to the lack of a role for CD8α(+) T cells in vaccine efficacy, they in turn point to a role for GM-CSF in reducing HSV-1 latency.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Neutralizing antibody titers in immunized mice. CD8α−/−, CD8β−/−, and WT mice were immunized i.p. with avirulent HSV-1 strain KOS as described in Materials and Methods. Three weeks after the third immunization, mice were bled and neutralizing antibody titers were determined by plaque reduction assays. Each bar represents the average neutralizing antibody titer from 10 serum samples for WT mice, 16 serum samples for CD8α−/− mice, and 8 serum samples for CD8β−/− mice. The error bars indicate the standard errors.
FIG 2
FIG 2
Virus titers in mouse eyes following ocular infection of immunized mice. The immunized mice described in the legend to Fig. 1 were ocularly infected with 2 × 105 PFU/eye of virulent HSV-1 strain McKrae. The presence of infectious virus in the eyes of immunized mice was monitored daily by collecting tear films from 20 eyes for WT mice, 32 eyes for CD8α−/− mice, and 28 eyes for CD8β−/− mice, as described in Materials and Methods. The error bars indicate the standard errors.
FIG 3
FIG 3
Quantitation of LAT RNA in TG of immunized mice. TG from the mice immunized as described in the legend to Fig. 1 were harvested on day 28 p.i. Quantitative RT-PCR was performed on the TG from each mouse. In each experiment, an estimated relative copy number of the HSV-1 LAT was calculated using standard curves generated from pGem5317. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 103 to 1011 copies of LAT and then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral LAT RNA in the TG. Each bar represents the mean ± SEM from 18 TG for WT mice, 32 TG for CD8α−/− mice, and 16 TG for CD8β−/− mice.
FIG 4
FIG 4
qRT-PCR analyses of the PD-1 transcript in the TG of latently infected mice. WT, CD8α−/−, and CD8β−/− mice were immunized i.p. with avirulent HSV-1 strain KOS or mock immunized as described in the legend to Fig. 1. Total RNA was isolated from each individual TG and used to estimate the relative expression of the PD-1 transcript in the TG of WT, CD8α−/−, or CD8β−/− mice. GAPDH expression was used to normalize the relative expression of each transcript in the TG of immunized mice. For mock-immunized mice, each bar represents the mean ± SEM from 20 TG, while for immunized mice, each bar represents the mean ± SEM from 18, 32, and 16 TG for WT, CD8α−/−, and CD8β−/− mice, respectively.
FIG 5
FIG 5
Expression of gB in the corneas (A) and TG (B) of infected mice. CD8α−/− and WT mice were immunized as described in the legend to Fig. 1 and ocularly infected as described in the legend to Fig. 2. gB expression in the cornea and TG was determined on days 3 and 5 p.i. In each experiment, an estimated relative copy number of HSV-1 gB was calculated using standard curves generated from pAC-gB1. Briefly, the DNA template was serially diluted 10-fold such that 5 μl contained from 103 to 1011 copies of LAT and then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of each transcript in the cornea and TG of infected mice. Each bar represents the mean ± SEM from 6 corneas or TG.
FIG 6
FIG 6
Expression of IFN-α in corneas (A), TG (B), and spleens (C) of infected mice. Total RNA isolated from individual mouse corneas and TG as described in the legend to Fig. 5 as well as RNA from the spleens of the same mice was used to estimate the relative levels of expression of IFN-α transcripts in WT and CD8α−/− immunized mice. IFN-α expression in the cornea, TG, and spleen was determined on days 3 and 5 p.i. GAPDH expression was used to normalize the relative expression of each transcript in the cornea, TG, or spleen in each group. Each bar represents the mean ± SEM from 6 corneas or TG and 3 spleens.
FIG 7
FIG 7
Expression of IFN-β in corneas (A), TG (B), and spleens (C) of infected mice. Total RNA isolated from individual mouse corneas and TG as described in the legend to Fig. 5 as well as RNA from the spleens of the same mice was used to estimate the relative levels of expression of IFN-β transcripts in WT and CD8α−/− immunized mice. IFN-β expression in the cornea, TG, and spleen was determined on days 3 and 5 p.i. GAPDH expression was used to normalize the relative expression of each transcript in the cornea, TG, or spleen in each group. Each bar represents the mean ± SEM from 6 corneas or TG and 3 spleens.
FIG 8
FIG 8
Expression of IFN-γ in corneas (A), TG (B), and spleens (C) of infected mice. Total RNA isolated from individual mouse corneas and TG as described in the legend to Fig. 5 as well as RNA from the spleens of the same mice was used to estimate the relative expressions of IFN-γ transcripts in WT and CD8α−/− immunized mice. IFN-γ expression in the cornea, TG, and spleen was determined on days 3 and 5 p.i. GAPDH expression was used to normalize the relative expression of each transcript in the cornea, TG, or spleen in each group. Each bar represents the mean ± SEM from 6 corneas or TG and 3 spleens.
FIG 9
FIG 9
Expansion of CD11c+ CD4+ cells after injection of mice with GM-CSF. WT mice were injected once with GM-CSF DNA or Flt3L DNA or mock injected, as described in Materials and Methods. At 2 weeks postinjection and prior to HSV-1 infection, the spleens of some of the injected mice were harvested, and single cells were prepared, stained with anti-CD3, anti-CD4, anti-CD8α, and anti-CD11c MAbs, and analyzed by flow cytometry. CD3-negative/CD11c-positive cells were gated on expression of CD8α and CD4 cells. A minimum of 104 events was acquired on a gate including viable cells. The mean percentages of CD3− CD11c+ CD4− CD8α+ or CD3− CD11c+ CD4+ CD8α− cells are shown for each treatment from two experiments.
FIG 10
FIG 10
Detection of LAT (A) and PD-1 (B) following GM-CSF or Flt3L injection. WT mice were injected 3 times with GM-CSF or Flt3L DNA prior to ocular HSV-1 infection. Injected mice were ocularly infected with HSV-1 strain McKrae, and quantitative RT-PCR was performed to assay LAT and PD-1 expression. Each point represents the mean ± SEM from 10 TG.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jordan MC, Jordan GW, Stevens JG, Miller G. 1984. Latent herpesviruses of humans. Ann. Intern. Med. 100:866–880. 10.7326/0003-4819-100-6-866 - DOI - PubMed
    1. Whitley RJ, Kimberlin DW, Roizman B. 1998. Herpes simplex viruses. Clin. Infect. Dis. 26:541–553. 10.1086/514600 - DOI - PubMed
    1. Dawson CR. 1984. Ocular herpes simplex virus infections. Clin. Dermatol. 2:56–66. 10.1016/0738-081X(84)90066-X - DOI - PubMed
    1. Barron BA, Gee L, Hauck WW, Kurinij N, Dawson CR, Jones DB, Wilhelmus KR, Kaufman HE, Sugar J, Hyndiuk RA, Laibson PR, Stulting RD, Asbell PA, for the Herpetic Eye Disease Study Group. 1994. Herpetic Eye Disease Study. A controlled trial of oral acyclovir for herpes simplex stromal keratitis. Ophthalmology 101:1871–1882 - PubMed
    1. Ghiasi H, Kaiwar R, Nesburn AB, Slanina S, Wechsler SL. 1994. Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice. J. Virol. 68:2118–2126 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-