A protective role for dengue virus-specific CD8+ T cells

Abstract

Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

FIGURE 1

DENV infection results in a CD8⁺ T cell response in wild-type and IFN-α/βR^-/- mice, but detectable levels of viremia only in IFN-α/βR^-/- mice. A, Wild-type mice (n = 3) infected with 10¹¹ GE of S221 and IFN-α/βR^-/- mice (n = 6) infected with 10¹⁰ GE were bled and the DENV RNA levels in the serum were measured by real-time RT-PCR. The dashed line indicates the limit of detection. B, Blood lymphocytes were obtained from wild-type mice (n = 4) on days 3, 6, 8, and 13 after infection with 10¹¹ GE of S221. The percentage of CD44^highCD62L^low cells (gated on CD8⁺ T cells) is indicated. C, The numbers of splenic CD8⁺ T cells in naive IFN-α/βR^-/- mice (n = 4) and IFN-α/βR^-/- mice infected with 10¹⁰ GE of S221 (n = 7) are shown. ***, p < 0.0001 for naive vs infected mice. D, Blood lymphocytes were obtained from IFN-α/βR^-/- mice (n = 3) on days 3, 5, 7, 10, and 13 after infection with 10¹⁰ GE of S221. The percentage of CD44^highCD62L^low cells (gated on CD8⁺ T cells) is shown.

FIGURE 2

Depletion of CD8⁺ T cells before DENV infection results in increased viral loads. IFN-α/βR^-/- mice were depleted of CD8⁺ T cells by administration of an anti-CD8 Ab (or given an isotype control Ab) 3 days and 1 day before infection with 10¹¹ GE of S221. Mice were sacrificed 6 days later, and DENV RNA levels in the serum, spleen, liver, and brain were quantified by real-time RT-PCR. Data are expressed as DENV copies per ml of sera or DENV units normalized to 18S rRNA levels for the spleen, liver, and brain. Each symbol represents one mouse, the bar represents the geometric mean, and the dashed line is the limit of detection. *, p < 0.001 for serum; ***, p < 0.0001 for spleen and brain; and p = 0.39 for viral load in the liver of CD8-depleted mice compared with control mice.

FIGURE 3

Identification of DENV-derived epitopes recognized by CD8⁺ T cells. IFN-γ ELISPOT was performed using CD8⁺ T cells isolated from wild-type mice 7 days after infection with 10¹¹ GE of S221. The data are expressed as the mean number of net SFC per 10⁶ CD8⁺ T cells. Three independent experiments performed in triplicate were averaged and the error bars represent the SEM. The criteria for positivity were net SFC per 10⁶ cells of ≥20, a stimulation index of ≥2.0, and p < 0.05 when compared with an irrelevant control peptide. The 12 positive peptides identified are shown.

FIGURE 4

Confirmation of DENV-derived CD8⁺ T cell epitope identification in wild-type and IFN-α/βR^-/- mice by ICS. A and B, Splenocytes harvested from wild-type (A) or IFN-α/βR^-/- (B) mice 7 days after infection with 10¹¹ or 10¹⁰ GE of S221, respectively, were restimulated in vitro with individual DENV peptides or an irrelevant peptide. Cells were then stained for surface CD8 and intracellular IFN-γ and analyzed by flow cytometry. The response to the irrelevant peptide was subtracted from the response to each DENV peptide, and the number of CD8⁺ T cells producing IFN-γ is indicated. Results are expressed as the mean ± SEM of 13 wild-type and 7 IFN-α/βR^-/- mice tested in at least three independent experiments. C and D, Kinetics of the DENV-specific CD8⁺ T cell response. Wild-type mice (C, n = 4) and IFN-α/βR^-/- mice (D, n = 3) were infected with 10¹¹ or 10¹⁰ GE of S221, respectively, and blood lymphocytes were isolated at various time points. Stimulation and ICS were performed as in A and B and the percentage of CD8⁺ T cells producing IFN-γ is shown.

FIGURE 5

DENV-specific CD8⁺ T cells have a polyfunctional phenotype. Splenocytes harvested from wild-type or IFN-α/βR^-/- mice 7 days after infection with 10¹¹ or 10¹⁰ GE of S221, respectively, were restimulated in vitro with C_51-59, NS4B_99-107, or an irrelevant peptide. An anti-CD107a Ab was added for the duration of the stimulation. Cells were then stained for surface CD8, intracellular IFN-γ and TNF-α, and analyzed by flow cytometry. Representative density plots are shown.

FIGURE 6

DENV-specific CD8⁺ T cells are cytotoxic and protective in vivo. A, In vivo killing of DENV peptide-pulsed cells. IFN-α/βR^-/- mice infected 7 days previously with 10¹⁰ GE of S221 were injected i.v. with CFSE-labeled target cells pulsed with C_51-9, NS2A_8-15, NS4B_99-107, NS5_237-245 or a pool of the four peptides (n = 3-6 mice/group). After 4 h, splenocytes were harvested, analyzed by flow cytometry, and the percentage killing was calculated. B, Peptide immunization results in enhanced DENV clearance. IFN-α/βR^-/- mice were immunized s.c. with 50 μg each of four DENV peptides (C_51-59, NS2A_8-15, NS4B_99-107, NS5_237-245) and 100 μg of helper peptide in IFA or mock immunized with 100 μgof helper peptide and DMSO in IFA. Mice were infected with 10¹¹ GE of S221 virus 12-13 days later and then sacrificed 4 days after infection. A separate group of peptide-immunized mice were depleted of CD8⁺ T cells before infection. DENV RNA levels in the serum were quantified by realtime RT-PCR. Each symbol represents one mouse; the bar represents the geometric mean, and the dashed line the limit of detection. ***, p < 0.0001 comparing peptide-immunized with mock-immunized mice; **, p < 0.001 comparing peptide-immunized with peptide-immunized/CD8-depleted mice.