Figure 4. CD1d^dep NKT cells accelerate DENV clearance and promote CD8⁺ T cell recruitment and activation in DENV-infected skin.

WT and CD1d-KO mice were infected with 2 × 10⁵ PFU of DENV2 s.c. by FP injection. (A) DENV NS3 staining of infected cells including DCs and monocyte/macrophages at day 3 in the dLNs of WT and CD1d-KO mice. Scale bars: 50 μM. Additional images are provided in Supplemental Figure 2. (B and C) DENV2 (E protein) gene copy numbers in the (B) FP skin and (C) dLNs were determined on days 3 and 5 after infection by qPCR. Single-cell suspensions from FPs and dLNs were stained with antibodies against CD3, CD4, CD8, γδTCR, and CD69 for characterization of the T cell response by flow cytometry. Total and activated CD8⁺ CTL populations in the (D) FP skin and (E) dLNs were determined on days 0, 3, and 5 after infection. Tracking of local versus recruited cells was performed by CFSE labeling prior to DENV2 infection. (F) CFSE⁺ (red) or CFSE^– (gray) CD8⁺ T cell (NK1.1^–CD3⁺CD8⁺) populations in the FPs were determined on day 3 after infection. To track infected cell migration, CFSE was injected into the FPs of WT and CD1d-KO mice 3 days after DENV2 infection. (G) DENV2-infected monocytes (mono) (CD45⁺CD11b⁺CD11c^–MHCII⁺DENV-NS3⁺CFSE^–; gray) and FP-derived, DENV2-infected monocyte/macrophage (mac) (CD45⁺CD11b⁺CD11c^–MHCII⁺DENV-NS3⁺CFSE⁺; orange) numbers in the dLNs were determined 5 days after infection. (H) DENV2-infected DCs (CD45⁺CD11b^–CD11c⁺MHCII⁺DENV-NS3⁺CFSE^–; gray) and FP-derived, DENV2-infected DCs (CD45⁺CD11b^–CD11c⁺MHCII⁺DENV-NS3⁺CFSE⁺; purple) in dLNs were enumerated 5 days after infection. (I) Total and activated CD4⁺ T cells in dLNs were determined on days 0, 3, and 5 after infection. For all panels, n = 6 mice for each time point. Data are represented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Tukey’s post test. For D–I, stacked bars are presented showing CFSE⁺ and CFSE^– populations. CD1d^dep NKT cells promote activation of CD8⁺ T cells in DENV-infected skin and clearance of DENV from skin and dLNs.