Memory CD8+ T cells from naturally acquired primary dengue virus infection are highly cross-reactive

Abstract

Cross-reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T-cell response to heterologous serotypes, we isolated HLA-A(*)1101-restricted epitope-specific CD8(+) T-cell lines from primary DENV-immune donors. Cell lines exhibited marked cross-reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross-reactivity between the DENV-1 and DENV-3 epitope variants. In vitro-stimulated T-cell lines consistently revealed a hierarchical induction of MIP-1β>degranulation>tumor necrosis factor α (TNFα)>interferon-γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose-dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP-1β production. This is the first study to show significant DENV serotype-cross-reactivity of CD8(+) T cells after naturally acquired primary infection. We also show qualitatively different T-cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.

FIGURE 1. Three predominant patterns of serotype-cross-reactivity in HLA-A*1101-restricted T cell lines

(A) Gating strategy used to identify tetramer⁺ cells. (B) Cell lines were stained with each of the three tetramer variants and reveal three types of serotype-cross-reactivity: pD1 serotype-specific, pD1-3/4 cross-reactive, pD1-2-3/4 cross-reactive. Cells were gated on live, CD3⁺ singlet lymphocytes. (C) ⁵¹Cr release assays demonstrate peptide dose-dependent cytolytic activity of representative cell lines at an E:T ratio of 10:1. NS = no stimulation.

FIGURE 2. Hierarchical response of effector functions in epitope-specific T cells

(A) Gating strategy and representative flow plots of a cell line in response to the absence or presence of agonist peptide. (B) pD1 serotype-specific, (C) pD1-3/4 cross-reactive, and (D) pD1-2-3/4 cross-reactive cell lines were stimulated with each of the peptide variants in intracellular cytokine staining assays. All possible combinations of the four effector functions (MIP-1β (M), TNFα (T), and IFNγ (I) production as well as degranulation (C), as determined by CD107a staining) are displayed across the x axis. Mean frequencies of responding CD8⁺ T cells of 10E11 (n = 2), 10C11 (n = 4), and 10B8 (n = 2) are shown. Peptide concentrations are shown in μg/mL.

FIGURE 3. Phosphorylation of ERK1/2 does not correlate with MIP-1β production

(A) Peptide dose-dependent phosphorylation of ERK1/2 after stimulation with epitope variants. (B) Average fold change in mean fluorescence intensity (MFI) of pERK1/2 in the cell lines 10E11 (n = 3), 10C11 (n = 4), and 10G5 (n = 2) after stimulation with decreasing concentrations (10, 1, 0.1, 0.01 μg/mL) of the three peptide variants. Fold change is relative to ‘no stimulation’ control.

FIGURE 4. pD2 induces a short-lived activation signal in a pD1-3/4 cross-reactive cell line

(A) Time course of pERK1/2 after pD2 stimulation of the cell line 10C11. One of 2 independent experiments is shown. (B) Phosphorylation of CD3ζ after stimulation of 10C11 with each of the three peptide variants. Histograms are shaded to emphasize the change in MFI. One of 3 independent experiments is shown. (C) Time course of CD137 expression after stimulation of 10C11 with pD1, pD2, and a control non-dengue HLA-A*1101-restricted viral peptide. The peptide concentration used for these experiments was 10μg/mL. NS = no stimulation.