Mapping of the CD4⁺ T cell response in the LysMCre⁺Ifnar1^fl/fl mouse model of primary ZIKV infection.

Five-week-old LysMCre⁺Ifnar1^fl/fl C57BL/6 mice were infected retro-orbitally with 10⁴ FFU of ZIKV strain MR766 or FSS13025 in 10% FBS/PBS or were mock-infected (10% FBS/PBS alone). Data are the mean ± SEM of n = 4–6 mice/group. (A–C) Splenocytes were removed on day 7 post-infection and analyzed for the percentage of (A) CD44⁺CD4⁺ T cells, (B) CD49d⁺CD11a⁺ T cells, and (C) granzyme B⁺CD4⁺ T cells. (D–F) Splenocytes were removed on day 7 post-infection and stimulated with the indicated ZIKV-derived peptides and brefeldin A. The percentage CD44⁺CD4⁺ T cells producing (D) IFNγ, (E) IFNγ and TNF, and (F) IL-2 was measured by ICS. Cells stimulated with DMSO or PMA/ionomycin served as negative and positive controls, respectively. (G) Summary of the data shown in (D–F). (H) In vivo killing of ZIKV peptide-pulsed target cells. LysMCre⁺Ifnar1^fl/fl mice were retro-orbitally mock-infected (n = 4) or infected with 10⁴ FFU ZIKV MR766 (n = 5) and FSS13025 (n = 4) for 7 days, and then injected retro-orbitally with naïve C57BL/6 splenocytes (n = 4) pulsed with a pool of ZIKV peptides (E_346-360, E_644-658, NS3_1740-1754, NS4B_2480-2494, NS5_2604-2618, NS5_2738-2752) or treated with DMSO. After 12 h, the splenocytes were harvested from recipient mice, analyzed by flow cytometry, and the percentage ZIKV-specific cytotoxicity was calculated. *P < 0.05, **P < 0.01 by the Mann–Whitney U test. The production of cytokines after stimulation with each peptide was compared to the negative control (DMSO) using one-way ANOVA t-test. **** P < 0.0001. Data are representative of two independent experiments.