Percp cd3e

The cells were stained for surface and intracellular/intranuclear, and analysis by FACS Canto flow cytometer (BD Biosciences). The following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD3, APC-CD3e, PerCP-CD3e, APC Cy7-CD3, PE-CD11b,PE Cy7-CD11b, FITC-MHC-II, PerCP Cy5.5-MHCII, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CD25, PE Cy7-CD25, PerCp Cy5.5-CD25, APC-CD138, FITC-CD40, PE-FoxP3, FITC-IL10 and anti-CD16/32 (Fc block) were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45⁺ cells. Acquired FCS files were analyzed by Flow Jo-V10. Treg cell population were identified based on CD25⁺FoxP3⁺ cell in CD3⁺CD4⁺ population. To observe the Treg cell population, CD4⁺ cells were analyzed for the expression of CD25 and FoxP3 and gated on the double positive population. Data were obtained from three to four independent experiments with 4–6 pairs of mice in each experiment. In each experiment, equal number (2/3 males and 2/3 females from each group) of male and female mice were used.