Cd4 gk1

Single-cell suspensions were prepared from the bone marrow of uninfected WT mice and intravaginal WT HSV-2 infected vaginal tissues of Oasl1^+/− or Oasl1^−/− mice. To obtain FACS-sorted Ly6C^high monocytes and CD4⁺ T cells, cells were stained with Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70), and CD45.2 (104) (BD Biosciences); CD3ε (145-2C11) and CD4 (GK1.5) (Tonbo biosciences) after incubating for 15 min on ice in the presence of anti-CD16/32 (2.4G2, Tonbo biosciences) antibody to block Fc receptors. Live cells were gated on the basis of DAPI exclusion. Stained cells were sorted by FACS Aria (BD Biosciences). Sorted vaginal Ly6C^high monocytes (CD45.2⁺Ly6G^-Ly6C^highCD11b⁺) and CD4⁺ T cells (CD45.2⁺CD3ε⁺CD4⁺) were more than 90% pure as assessed by post-sort analyses.

Viability dye was purchased from Invitrogen (catalog 65-0865-14). The following mAbs were purchased from Invitrogen: CD107a (ebio1D4B, catalog 50-1071-82), CD107b (ebioABL-93, catalog 50-1072-82), Isotype IgG2aκ (eBR2a, catalog 50-4321-80), IFN-γ (XMG1.2, catalog 45-7311-82), TNF (MP6-XT22, catalog 12-7321-82), CD11b (M1/70, catalog 45-0112-82), and CD11c (N418, catalog 11-0114-85). From Tonbo, we purchased CD4 (GK1.5, catalog 60-0041-U100). From BioLegend, we purchased CD8 (53-6.7, catalog 100725). From eBioscience, we purchased CD45 (30-F11, catalog 48-0451-82).
Intracellular cytokine staining was performed as previously described (84 (link)). Briefly, live splenocytes from experimental mice were cocultured 1:1 with freshly isolated antigen presenting cells (APCs; negative fraction of EasySep Mouse 90.2 positive isolation kit II, Stemcell Technologies, catalog 18951) from naive donor mice at 37°C overnight. After 18 hours of incubation, Golgiplug (BD Biosciences, catalog 555029) was added to the culture wells for 4 hours. After surface staining, cells were subsequently fixed/permeabilized (eBioscience, catalog 00-5523-00) and stained for intracellular cytokines per the manufacturer protocols.