Epigenetically altered T cells were identified with PE-anti-Kir2DL4/CD158D (clone 181703; R&D Systems, Minneapolis, MN), anti-CD40L-PE (clone hCD40L-M91), anti-CD11a-PE (clone HI111), PEanti-CD70 (clone Ki-24), and PE-Cy5-anti-CD4 (clone RPA-T4) (Becton Dickinson, Franklin Lakes, NJ). PE-anti-CD158b (clone CHL), PE-anti-CD158i (clone FES172), PE-anti-CD158b1/b2, j (clone GL183), and PE-anti-CD158a, h (clone EB6B) were from Beckman Coulter (Brea, CA) and analyzed by multicolor flow cytometry as described [31 (link)]. All antibodies were titrated to determine their optimal concentrations prior to use.
CD4+GFP+ cells were identified by gating on GFP then analyzed for KIR, perforin, CD40L, and CD70. For sorting, the cells were also stained with (4′, 6-diamidino-2-phenylindole) (DAPI), and live transfected cells (CD4+GFP+DAPI−) were isolated prior to analysis by RT-PCR.
CD4+GFP+ cells were identified by gating on GFP then analyzed for KIR, perforin, CD40L, and CD70. For sorting, the cells were also stained with (4′, 6-diamidino-2-phenylindole) (DAPI), and live transfected cells (CD4+GFP+DAPI−) were isolated prior to analysis by RT-PCR.