Astrocyte–immune cell co-cultures

Naive CD4⁺ T cells and NK cells were isolated from the spleens of B6.SJL-Ptprca Pepcb/BoyJ mice (The Jackson Laboratory, 002014) or Ifng^EYFP reporter mice using Miltenyi beads (130-104-453 and 130-115-818). CD4⁺ T cells were activated overnight with 10 μg ml⁻¹ anti-CD3 (bioXcell, BE0001-1-A005mg) and 0.25 μg ml⁻¹ anti-CD28 (bioXcell, BE0015-5-A005mg). For co-culture with EAE T cells, T cells were isolated from mice undergoing EAE at the peak of disease by flow cytometry based on CD3, CD4, and DR5 expression. DR5⁺ and DR5⁻ T cells were isolated separately for co-culture. Astrocytes and CD4⁺ T cells cells were co-cultured in a 2:1 ratio. To block TRAIL, astrocytes were treated with IFNγ overnight, washed extensively with 1× PBS, and 2.5 μg ml⁻¹ of anti-TRAIL antibody (Thermo Fisher Scientific, 16-5951-85) or isotype control antibody (Thermo Fisher Scientific, 16-4321-81). Astrocytes were incubated with antibodies for one hour before addition of activated CD4⁺ T cells. In another set of experiments CD4⁺ T cells were activated with anti-CD3 and anti-CD28 as described above for 72 h and NK cells were activated with 100 ng ml⁻¹ LPS (Invivogen, tlrl-3pelps) for 48 h. Ifng^YFP+ and Ifng^YFP− cells were sorted and co-cultured with astrocytes. After 8 h of co-culture, cells were collected for FACS. Cell-surface antibodies were added for 30 min on ice, cells were washed and fixed to detect active-caspase-3 as described above. For TUNEL (Abcam, ab66108) staining, cells were fixed in 1% v/v PFA for 15 min on ice, washed with 1× PBS and incubated with 70% ethanol for 30 min on ice and washed twice. Cells were stained with staining solution (10 μl of reaction buffer, 0.75 μl of TdT enzyme, 8 μl of FITC-dUTP and 32.25 μl of water) for 60 min at 37 °C, washed and acquired on LSR Fortessa (BD Biosciences) or Symphony A5 (BD Biosciences). Antibodies used were: BV711 anti-mouse CD45.1 (BD Biosciences, 747742, 1:100), PE/Dazzle594 anti-CD11b (Biolegend, 101256, 1:100), PE anti-mouse CD3 (Biolegend, 100206, 1:100), BV650 anti-mouse CD4 (Biolegend, 100546, 1:100), PerCP-Vio700 anti-mouse DR5 (Miltenyi, MD5-1-3C2, 1:10), APC-Cy7 anti-IFNγ (BD Biosciences, 561479, 1:100), PE anti-mouse IL-17A (eBioscience, 12-7177-81, 1:100), BUV395 anti-mouse LAMP1, PE-Cy7 anti-mouse TRAIL, BUV661 anti-mouse CD45, BV786 anti-mouse CD11b, BV421 anti-mouse NK1.1 and BV750 anti-CD3.