Soluble cd3 clone cd3 2

GrzB ELISpot’s were performed in accordance with manufacturer instructions (Mabtech). 1×10⁵ memory CD4 and memory CD8 T cells were cultured (200 μl/well) for 24 hrs in 96-well flat-bottomed nitrocellulose plates (MultiScreen-HTS, Millipore). Wells were first prepared by treating with 70% ethanol for 2 mins and rinsing with sterile H₂0. Wells were then coated overnight at 4°C with 15 μg/ml GrzB mab (clone GB10). After GB10 coating, wells were washed with PBS, and pre-incubated with cell culture RPMI-1640 medium (200 μl/well) for 30 mins at room temp. Cells were then added to appropriate wells and either unstimulated (UT) or costimulated with 1 μg/ml soluble CD3 (clone CD3-2, Mabtech) + soluble CD28 mabs (clone CD28.2, BD Biosciences) for 48 hrs wrapped in aluminum foil at 37°C + 5%CO₂. After the treatment period, cells were gently harvested for flow cytometry staining, and wells were washed with PBS. 1 μg/ml biotinylated GrzB detection mabs (clone GB11) were added for 2 hrs at room temp. Wells were washed and streptavidin-HRP added for 1 hr. Wells were washed and TMB substrate added until spot development (10-30 mins). Color development was stopped with H₂0, and plates were air-dried, wrapped, and stored at room temp. until spot analysis. Spots were analyzed using CTL-ImmunoSpot S4 analyzer (Cellular Technology Limited).

Human memory CD4+CD45RO+ and memory CD8+CD45RO+ T cells were purified from PBMC of healthy buffy coat donors. PBMC were first isolated from buffy coats (Gulf Coast Regional Blood Center, Houston, TX) by using Ficoll-Paque PLUS (GE Healthcare). Memory CD4 and memory CD8 T cells were then negatively selected from PBMC with magnetic bead-based EasySep kits (Stemcell Technologies). Purities of memory CD4+CD45RO+ T cells were usually >90% and memory CD8+CD45RO+ T cells >80% as determined by flow cytometry. Natural killer cells were purified from PBMC with negative selection kits (Stemcell Technologies), and CD3-CD56+ purity was >90%. Cells were cultured at 37°C + 5%CO₂ in complete RPMI-1640 medium, supplemented with 10% FBS, 0.1 mM MEM nonessential amino acids, 2mM sodium pyruvate, 2mM HEPES, 1× antibiotic-antimycotic and 2mM L-glutamine.
Most experiments involved no stimulation (UT) or stimulation of 1-5×10⁵ memory CD4 and memory CD8 T cells, or NK cells (200 μl/well of 96-well flat-bottom plates, or 1 ml/well of 24-well plates, as indicated) for 24 hrs with 1 ml/ml coated CD3 (clone UCHT1, BD Biosciences) or soluble CD3 (clone CD3-2, Mabtech) + 1 μg/ml CD28 (clone CD28.2, BD Biosciences) mabs. For some experiments, cells were stimulated with 100 ng/ml recombinant IL2 (Biolegend) or soluble CD3 mab alone for 24 hrs with or without brefeldin (GolgiPlug, BD Biosciences).