Dengue-specific CD4⁺ T-cell responses in primary and secondary dengue virus (DENV) infections. (A and B) Intracellular IFN-γ staining was performed on splenocytes from mice that were infected with primary DENV-1, primary DENV-2, primary DENV-3, or primary DENV-2 followed by secondary DENV-1 or DENV-3. CD4⁺ T-cell responses to whole virus antigens were measured. The frequencies from cells stimulated with control Vero cell antigen were subtracted from those stimulated with viral antigen. (A) Representative IFN-γ staining in response to inactivated DENV antigens. The bottom number in each panel represents the frequency of the IFN-γ response with the media background subtracted, and the top number represents the frequency of the IFN-γ response with the Vero antigen control subtracted. (B) Comparison of CD4⁺ T-cell responses in primary versus secondary dengue virus infections. Bars show medians and 95% confidence intervals. P values were calculated by the Mann-Whitney test. Asterisks denote responses with significant differences between immunization groups by ANOVA (p < 0.05); post-hoc testing showed that mice with secondary DENV-1 infection had significantly higher responses than the primary DENV-1, DENV-2, or DENV-3 infection groups for all antigens, and significantly higher responses than the secondary DENV-3 infection group for DENV-2 antigen. These data were generated in three individual experiments (n = 8 mice per group). (C–F) Bulk culture cell lines were generated from splenocytes from mice that were administered DENV-2 28 d earlier. Cells were stimulated with inactivated DENV-1 (circles) or DENV-3 (diamonds) antigens. Day 7 (C and D) and day 14 (E and F) post-stimulation, cells were tested for IFN-γ production to whole virus antigen for all four serotypes by intracellular cytokine staining. (C) Representative intracellular cytokine staining of bulk culture CD4 T cells on day 7. The bottom number in each panel represents the frequency of the IFN-γ response with the media background subtracted, and the top number represents the frequency of the IFN-γ response with the Vero antigen control subtracted. (D) Cross-reactivity of memory CD4 T-cell responses from DENV-2–immune mice on day 7. The median responses are represented by the black bars. (E) Representative intracellular cytokine staining of bulk culture CD4 T cells on day 14. The bottom number in each panel represents the frequency of the IFN-γ response with the media background subtracted, and the top number represents the frequency of the IFN-γ response with the Vero antigen control subtracted. (F) Cross-reactivity of memory CD4 T-cell responses from DENV-2–immune mice on day 14. The median responses are represented by the black bars. P values were calculated by the Mann-Whitney test (N = 4 individual cultures from individual animals per group for D; n = 3 individual cultures from individual animals per group for F).

Transfer of memory CD4⁺ T cells affects the CD8⁺ T-cell response to secondary dengue virus (DENV) infections. CD4⁺ and CD8⁺ T cells were isolated from DENV-2-immune mice. The CD8⁺ T cells alone or in combination with the CD4⁺ T cells were transferred IV to naïve mice. The following day, the mice were administered either DENV-1 or DENV-3. Splenocytes were harvested on day 9 post-infection. IFN-γ responses to CD8⁺ T-cell epitopes D1/3 NS3 and D2/4 NS3 were measured by intracellular cytokine staining. (A) Representative IFN-γ staining in response to peptides D1/3 NS3 and D2/4 NS3. The number in each panel represents the frequency of the IFN-γ response with the media background subtracted. (B) Comparison of CD8⁺ T-cell responses after adoptive transfer of memory CD8⁺ cells alone or both memory CD4⁺ and CD8⁺ T cells. Symbols show individual mice; medians are represented by the horizontal bars. P values <0.1 calculated by the Mann-Whitney test are shown. P values <0.05 are considered significant. These data were generated from three individual experiments (n = 6 per group).