Figure 4

Adaptive immune responses. (a,b) viSNE analyses of T cell activation in rhesus (a) and cynomolgus (b) macaques, as measured by CD69 expression. Dot plots are concatenated for CD8-depleted and nondepleted animals within each species. The viSNE clustering profiles of CD4 and CD8 T cell subsets (left), including naïve, central memory (CM), and effector memory (EM) T cells correspond to cell populations in the CD69 heatmaps in nondepleted and CD8-depleted animals at 1 and 10 dpi (right). Black ovals indicate EM CD4 populations in CD8-depleted animals at 10 dpi, which showed higher levels of CD69 expression compared to nondepleted animals at the same timepoint. (c) Proliferation of EM CD4 T cells by Ki67 expression. Area-under-the-curve analysis revealed a significant difference in EM CD4 T cell proliferation among CD8-depleted (n = 4) and nondepleted (n = 5) animals by a Mann–Whitney test (p = 0.0159). viSNE plots were generated using viSNE software (Cytobank version 7.0, https://www.cytobank.org/platform.html). (d) CD4 T cell responses in rhesus macaques, assessed by intracellular cytokine staining (ICS) of PBMCs stimulated with viral peptides derived from the indicated ZIKV proteins (C = capsid; M = membrane; E = envelope; NS1 = nonstructural protein 1, consistent throughout). CD4 T cell responses were identified by co-positivity for IL-2 and IFNγ. (Inset): representative antigen-specific cytometry plots for R64357 (CD8-depleted) at 30 dpi. Dot plots were generated using FlowJo (version X.10.4.2, https://www.flowjo.com/). (e) CD8 T cell responses, determined by ICS for perforin and IFNγ co-positivity. (f) Serum neutralizing antibody titers in rhesus macaques, represented as PRNT90.