Effective Priming of Herpes Simplex Virus-Specific CD8+ T Cells In Vivo Does Not Require Infected Dendritic Cells

Abstract

Resolution of virus infections depends on the priming of virus-specific CD8⁺ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8⁺ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8⁺ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8⁺ T cell priming during a peripheral virus infection.IMPORTANCE The ability of some DC to present viral antigen to CD8⁺ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo Our finding that HSV-specific CD8⁺ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8⁺ T cell priming during a peripheral virus infection.

Keywords: CD8 T cells; HSV-1; cross-presentation; dendritic cells; direct presentation.

FIG 1

Irf8-dependent cells, but not pDC, are required for HSV-specific CD8⁺ T cell priming. (A) Representative plots of DC and DC subsets enriched from the brachial LN of Irf8^−/− and WT mice. (B to D) Absolute number of gB_498–505-specifc CD8⁺ T cells (B), frequency of IFN-γ producing CD8⁺ T cells in the spleen (C), and virus titers in the skin (D) of Irf8^−/− and WT mice infected with HSV-1 on flank skin 3 and 7 days earlier. All data are pooled results from at least two independent experiments (n = 5 per experiment) and are expressed as mean + standard error of the mean (SEM). (E) Representative plots of splenic pDC in BDCA2-DTR mice treated with or without DTX and absolute number of gB_498–505-specific CD8⁺ T cells in the spleens of BDCA2-DTR mice treated with or without DTX that were infected with HSV-1 on flank skin 7 days earlier. Asterisks indicate statistically significant differences versus controls as assessed by the Student t test (***, P < 0.0001).

FIG 2

Langerin-positive DC, but not LC, are required for HSV-specific CD8⁺ T cell priming. (A) Schematic depicting the DTX treatment regimen for the assessment of DC depletion. Mice were treated with DTX on days −4 and −2. DC in the brachial LN and the epidermis were analyzed 2 days or 21 days after the last DTX treatment. (B) Representative plots of MHC-II^hi CD11c^hi cells enriched from the brachial LN on day 2 or day 21 after DTX treatment on two consecutive days. Cells were first plotted based on expression of CD8 and GFP (langerin) to identify CD8⁺ DC. The CD8⁻ fraction was then further divided into LC (GFP⁺ CD103⁻) and CD103⁺ DC (GFP⁺ CD103⁺). (C and D) Top panels, schematics depicting the DTX treatment regimen to determine its impact on HSV-specific CD8⁺ T cell priming. Mice were treated with DTX on days −4 and −2. On day 0 (C) or day 21 (D) mice were infected on the skin with HSV-1, and the spleens were analyzed for virus-specific CD8 T cells 7 days later. Bottom panels, absolute numbers of gB_498–505-specific CD8⁺ T cells in the spleen on day 7 after HSV-1 skin infection of Lg-DTR mice that last received DTX 2 days (C) or 21 days (D) earlier relative to those in PBS controls. All data are pooled results from at least two independent experiments (n = 3 to 5 per experiment) and are expressed as mean + SEM. Asterisks indicate statistically significant differences versus controls as assessed by one-way analysis of variance (ANOVA) (*, P < 0.05).

FIG 3

No evidence for virus infection of DC in the LN draining the site of infection in wild-type mice. (A) Viral titers in homogenates of the brachial LN measured 2 days after epidermal infection of Ifnar2^−/− and WT mice with HSV-1. (B) Representative plots of MHC-II^hi CD11c^hi DC enriched from the brachial LN of Ifnar2^−/− and WT mice infected 2 days earlier with a GFP-expressing HSV-1 strain. (C) Relative expression of viral mRNA in DC subsets isolated from the brachial LN of Ifnar2^−/− and WT mice infected with HSV-1 on the skin 2 days earlier. Data are pooled results from two independent experiments (n = 5 per experiment) and are expressed as mean + SEM. N.D., not detectable.

FIG 4

R-DTA mice inoculated with HSV-Cre eliminate infected cells in the DRG and DC in the LN. (A to C) Photograph of the primary- and secondary-site viral lesions (A) and viral titers (B and C) in wild-type and R-DTA mice infected on the skin with a Cre-expressing HSV strain compared to infection of WT mice with a TK-deficient HSV strain. (D) Percentage of GFP⁺ DC in the brachial LN of WT, Ifnar2^−/−, R-DTA × Ifnar2^−/−, and R-DTA mice infected 2 days earlier on the skin with HSV-Cre-GFP. Data are pooled from two independent experiments (n = 5 per experiment) and are expressed as mean + SEM. N.D., not detectable.

FIG 5

Ex vivo CD8⁺ DC presentation of HSV-derived antigen and HSV-specific CD8⁺ T cell priming does not require directly infected DC. (A and B) Absolute numbers of proliferated, CTV-labeled gBT-I upon coincubation for 60 h with serial dilutions of LN DC subsets isolated 2 days after skin HSV-Cre infection of WT (A) and R-DTA (B) mice. (C and D) Absolute number of gB_498–505-specific CD8⁺ T cells (C) and frequency of IFN-γ-producing CD8⁺ T cells (D) in the spleens of R-DTA (open bars), WT (black bars), and R-DTA × Ifnar2^−/− mice (gray bars) infected with HSV-Cre or HSV-ΔTK on flank skin 7 days earlier. Data are pooled from three independent experiments (n = 5 to 10 per group and experiment) and are expressed as mean + SEM.