Anti helios clone 22f6

For cell analysis from primed mice, spleens were dissociated into single-cell suspensions and RBCs were lysed. For the CNS leukocytes, single-cell suspensions were prepared as previously described (21 (link)) from spinal cords of individual mice perfused with 20 ml of PBS. For B7-H4Ig binding analysis, cells were collected and treated with Cytochalasin D (Sigma-Aldrich, St. Louis, MO) for 2 h at 37°C. The cells were washed in PBS, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies, Grand Island, NY), and blocked with anti-CD16/32 (eBioscience) prior to incubating the cells with either directly conjugated Control Ig or hB7-H4Ig. Following incubation with Control Ig or hB7-H4Ig, the cells were washed and then stained with the indicated Abs: anti-CD3 (mouse clone 145–2C11; human clone OKT3), anti-CD4 (mouse clone RM4–5; human clone OKT4), anti-Foxp3 (clone FJK-16s), anti-Helios (clone 22F6) (eBioscience), anti-CD25 (clone PC61) (eBioscience), anti–Nrp-1 (mouse polyclonal sera; human clone no. 446921), anti-PlxnA4 (mouse clone no. 707201; human clone no. 707206), and anti-Sema3a (mouse and human clone no. 215803) (R&D Systems). A total of 10⁶ viable cells were analyzed per individual sample using a BD Canto II cytometer (BD Biosciences), and the data were analyzed using FlowJo version 9.5.2 software (Tree Star, Ashland, OR).