αcd3 cd2 cd28 t cell activation beads

Primary human keratinocytes and fibroblasts were cultured to confluence in 96-well flat-bottomed plates. T cells from the three sorted memory subtypes were stimulated with αCD3/CD2/CD28 T cell activation beads (Miltenyi Biotec, San Diego, CA) at a 1:10 ratio for 24 hr. Stimulated memory T cells from each subtype were added to designated wells of confluent layers of keratinocytes, fibroblasts, or tissue culture wells without keratinocytes or fibroblasts (plastic) in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS and L-glutamine. At indicated time points, T cells were collected and immunostained with directly conjugated anti-human monoclonal antibodies from BioLegend and Life Technologies. Acquisition of flow cytometry samples was performed on a FACS Canto instrument and data were analyzed using FACS Diva software V8.0 (BD Biosciences).

Primary human keratinocytes were cultured to confluence in 96-well flat-bottomed plates. PBMC were stimulated with αCD3/CD2/CD28 T cell activation beads (Miltenyi Biotec, San Diego, CA) at a 1:10 bead:T cell ratio for 24 hours. PBMC were added to wells of keratinocytes in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS and L-glutamine (control medium) or in control medium plus rapamycin (Selleck Chemicals, Houston, TX) at indicated concentrations. Some wells cultures were started in control medium and switched to rapamycin containing medium on day 4 or 8. Cultures were fed with control or rapamycin containing medium every other day for 7 or 14 days. At the end of the culture period, T cells were collected and stained with directly conjugated anti-human monoclonal antibodies from BioLegend and Life Technologies.