T. gondii Antigen Stimulation Assay

Flow cytometry was performed on splenocytes and MLN cells as previously described [17 (link)]. Splenocytes (1 × 10⁶ cells/mouse) and MLN cells (1 × 10⁵ cells/mouse) were stimulated with T. gondii ME49 antigen (2 μg/mL) at 37 °C with 5% CO₂ for 2 h. Stimulated cells were stained with fluorophore-conjugated antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Invitrogen (Waltham, MA, USA). CD4⁺ T cells, CD8⁺ T cells, and germinal center B (GC B) cell populations were measured using the following antibodies: CD3 (PE- Cy7), CD4 (FITC), CD8 (PE), GL7 (PE), and B220 (FITC). All staining procedures were performed according to the manufacturer’s protocol. Stained cells were acquired by Accuri C6 flow cytometer and analyzed with the C6 Accuri software (BD Biosciences, Franklin Lakes, NJ, USA). For GC B cells, B220⁺ lung cells and splenocyte populations were gated, and from this population those positive for GL7 were gated. For CD4⁺ and CD8⁺ T cells, CD3-positive single cell populations were gated and from this population CD4⁺ and CD8⁺ cells were determined.