Further characterization of CD8 Treg
FoxP3 is a transcription factor responsible for regulatory activity. Since a number of molecules have been described to express on CD8 Treg (20-23), we used several of described markers on FoxP3+ and FoxP3- CD8+ T cells to further characterize CD8 Treg. Anti-CD3/CD28 activated purified CD8+ T cells were stained with following monoclonal antibodies and isotype controls: anti-CD8-PerCP-Cy5.5, anti-CD183-BV420, anti-CD25-PE-CF595, (all from BD Bioscience, San Diego, CA), anti-ICOS-APC, anti-CTLA-4-APC (from Biolegend, San Diego, CA). Cells were fixated and permeabilized using the Human FoxP3 Buffer Set from BD PharmingenTM (San Diego, CA), according to manufacturer’s instructions. After wash, the cells were stained with anti-FoxP3-PE and anti-LAP-1-PerCP-Cy5.5 for 30 minutes at room temperature. Cells were gated for FoxP3 and then examined for the expression of other markers on FoxP3+ and FoxP3- CD8+ T cells. Data in Figure 3A (% of positive cells) and Figure 3B (mean fluorescence intensity, MFI) show that FoxP3+ CD8+ T cells expressed significantly higher levels of CD25, CD278 (ICOS), CTLA-4, CD183, and LAP-1 (%) as compared to FoxP3- CD8 T cells. Therefore, we examined regulatory activity of sorted CD183+CD25highCD278+CD8+ Treg on B cell proliferation and immunoglobulin production.