Pe anti mice cd4

Spleens were collected from mice on day 28 post-prime immunization and were grounded in a 35 mm Petri dish containing 5 mL of mice 1× lymphocyte separation medium (DAKEWE, Shenzhen, China); suspensions were filtered using 40-μm filters into a new tube and were centrifuged at 800× g for 30 min. The lymphocyte layer was collected and washed with RPMI 1640 medium(Invitrogen, Carlsbad, CA, USA), and cell counts were stained with 0.4% trypan blue and counted using the Countess Automated Cell Counter system (Life Technologies, Carlsbad, CA, USA). Splenocytes were used for subsequent flow cytometry analysis, enzyme-linked immunospot assay (Elispot), and splenocyte proliferation assays. For flow cytometry, splenocytes (5 × 10⁶ cells in 6-well cell culture plates) were stimulated with 10 μg/mL VLPs or inactivated BVDV in the presence of 3 μg/mL Brefeldin A (eBioscience, San Diego, CA, USA) for 6 h at 37 °C and 5% CO₂, and cells were washed using staining buffer (eBioscience, San Diego, CA, USA) and stained with PE/cyanine7 anti-mice CD3 (BioLegend, San Diego, CA, USA), PE anti-mice CD4 (BioLegend, San Diego, CA, USA), and PerCP/cyanine5.5 anti-mice CD8a (BioLegend, San Diego, CA, USA). Cells were analyzed using BD Fortessa (BD Biosciences, CA, USA).

Fresh human PBMCs were stained with monoclonal antibodies, such as PercP-Cy5.5-anti-human CD3, APC/Cy7-anti-human CD4, PE-anti-human CD25, and Brilliant Violet 510-anti-human CD127 (all purchased from BioLegend, San Diego, CA, USA), in 0.5% BSA + 2 mM EDTA in PBS. Splenocytes were obtained from the spleens of mice by mechanical disruption and through a 200-gauge steel mesh. Splenocytes were lysed with 1_X red blood cells (RBCs) lysis buffer to remove RBCs. First, magnetic microbeads (Miltenyi Biotec) bead-based enrichment of CD4⁺T cells was performed. Then, cells were stained with monoclonal antibodies, such as PE-anti-mice CD4, APC/Cyanine7-anti-mice CD25, and Alexa-Fluor647-anti-mice CCR6 (all purchased from BioLegend), in 0.5% BSA + 2 mM EDTA in PBS. Human Treg cells and murine Treg cells were sorted using a SH800S cell sorter (SONY, Japan) and analyzed with the SH800 software (SONY, Japan). Human Treg cells were gated as CD3⁺CD4⁺CD25⁺CD127^- within the lymphocyte gate, which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). Mice Treg cells were gated as CD4⁺CD25⁺ within the lymphocyte gate which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). The purity of the sorted cell population was above 95% for human and 91% for mice, which was verified using post-sorting flow cytometry.