Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells

Abstract

The role of CD8(+) T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8(+) responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8(+) T cells is associated with protection from dengue virus disease.

Fig. 1.

HLA coverage and T-cell reactivity of the study population. (A) HLA allele coverage in the Sri Lankan cohort is shown. Bars represent the relative number of donors in which the donor-specific HLA alleles have been exactly matched (filled bars) or matched within the same supertype (open bars) with the 27 alleles selected for our study. The black line represents the cumulative number of donors in which 1–4 alleles have been matched exactly. (B) T-cell reactivity in blood donors after primary (n = 55; open bars) and secondary infection (n = 127; filled bars) with DENV. Shown are the relative response frequency (Left), the average magnitude of responses per donor (Center), and the average number of epitopes per donor (Right).

Fig. 2.

Immunodominant regions of the DENV polyprotein. (A) The genomic position of DENV-encoded proteins (C, capsid; prM, premembrane; M, membrane; E, envelope, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and the total observed response magnitude for every amino acid along the proteome is shown (filled bars). The data are expressed as total number of IFNγ SFCs per 10⁶ PBMCs. The heat map indicates the number of donors that showed a positive cytokine response to peptides within these regions. (B) Identified antigenic regions were plotted as a function of the percentage of the total response. Lines indicate the number of regions needed to account for 50%, 75%, and 90% of the total response, respectively.

Fig. 3.

Comparison between responses to conserved and serotype-specific epitopes. Representative donors who had been secondarily infected previously were incubated with donor-specific peptide pools (1μg/mL) originated either from regions serotype-specific for DENV3 (open circles) or regions conserved between two or more other serotypes (filled circles) for 6 h in the presence of brefeldin A. Cells were then stained with mAB against surface markers CD3, CD8, CD45RA, and CD27 and mAB against intracellular CD107a, IFNγ, TNFα, and IL2. Magnitude of response (A) and phenotype of responding cells (B) of the individual donors (n = 7) based on gating of the IFNγ producing cells are shown. The average CD45RA, CCR7, CD27, and CD107a expression for all responding cells is shown in C. Multifunctional responses are shown for individual donors (D) and as the average of all donors studied (E, n = 6). Avidity of responding T cells was determined by incubating PBMCs with ascending concentrations of peptide pools (0.001, 0.01, 0.1, 1, and 10 μg/mL). The peptide concentration necessary to induce 50% of the maximum responses (EC₅₀) was calculated, and the average EC₅₀ was compared between the conserved and serotype-specific epitopes (F).

Fig. 4.

HLA-linked T-cell responses. (A) Differential frequency and magnitude of HLA restricted responses in dengue-seropositive donors (n = 182). Frequency (filled bars) and the magnitude (open bars) of T-cell responses (as total SFCs per 10⁶ PBMC values) sorted according to their restriction element are shown. (B) Association of HLA restricted T-cell responses with disease susceptibility. Disease susceptibility was correlated with the average magnitudes of HLA restricted responses (Left), with the frequency and the magnitude of T-cell responses per donor (Center) and with the magnitude per epitope as well as breadth of response (Right). One-tailed Spearman test was then used to calculate correlations. The best positive correlation was observed with the magnitude of response per epitope (P = 0.02). (C) Multifunctionality of HLA restricted responses. Representative donors were stimulated with HLA restricted donor-specific peptide pools for 6 h in the presence of BFA. Cells were then stained with mAB against surface markers CD3 and CD8 and mAB against intracellular IFNγ, TNFα, and IL2. Pie charts represent cytokine profiles of individual donors. The relative number of cells producing one (dark gray areas), two (light gray areas), or three (white areas) of the measured cytokines is shown. Percentages in the pie charts represent the relative number of cells that produce two or more cytokines. Numbers under the pie charts represent the T-cell responses (as IFNγ SFCs per 10⁶ PBMCs) of this specific donor/allele combination in the ELISPOT assay (n = 4).