Isolation and Activation of CD4+ T Cells

Blood Leuko Paks from women were obtained from our IRB-approved collection facility at Dartmouth-Hitchcock Medical Center. CD4+ T cells were purified with the CD4+ T cell isolation kit (Miltenyi Biotech) following isolation of peripheral blood mononuclear cells (PBMC) by standard Ficoll density gradient centrifugation^{35 (link),56 (link)}. Freshly isolated blood CD4+ T cells were activated in vitro using Xvivo 15 media with Phenol Red plus Phytohemagglutinin (PHA, 2.5 µg/ml; Sigma, St Louis, MO) and IL-2 (50 U/ml, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann- La Roche Inc.) for 24hr as described previously^{56 (link)}. Activated CD4+ T cells were plated at a density of 1 × 10⁵ cells per well in round bottom ultra-low attachment 96-well culture plates (Corning, Corning, NY) in 0.2 ml of Immune Cell Media consisting of X-VIVO 15 Media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped human AB serum (Valley Biomedical, Winchester, VA) prior to treatment.