Fig. 5: Vaginal tissue Tregs differentially express Granzyme B after HSV-2 infection.

FoxP3^GFP mice were administered Depo provera s.c. and infected 5–7 days later ivag with WT HSV-2; controls received Depo provera only. On day 7 post-infection, VT and dLN were harvested and prepared for FACS to isolate CD4+ FoxP3+ Tregs. Single-cell RNA-sequencing was performed on dLN and VT Tregs and analyzed with the Seurat pipeline in R. n = 5 HSV-2+ and n = 3 uninfected. a UMAP visualization of graph-based clustering of VT and dLN Tregs from infected mice and dLN Tregs. b Heatmap showing the top 20 DEG in VT and dLN Tregs. Significant DEG were determined by log2FC > 0.5 and FDR < 0.01. A full DEG list can be found in Supplementary Table 4. c Violin plots of GzmB and Nkg7 expression in Treg populations. d, e C57BL/6 J mice were administered Depo provera s.c. in the neck ruff and infected ivag 5–7 days later with HSV-2. On days 0 (uninf.), 3, and 7 post-infection, dLN and VT were harvested for flow cytometry analysis. Tregs and CD8 T cells were identified as CD4+ FoxP3+ and CD8+, respectively. Representative granzyme B (GzmB) ex vivo staining in dLN (grey) and VT (blue) in d Tregs and e CD8 on days 0 and 7 post infection (left), quantified as a percentage of total Treg or CD8+ and by absolute number (right). n = 5 HSV-2+ and n = 5 uninfected. Data representative of three replicate experiments. Significance defined by paired t test or Kruskal–Wallis test followed by Dunn’s multiple comparison test; p < 0.05.