Isolation and Activation of CD4+ T Cells

Following removal of dead cells, CD4+ T cells were isolated by negative magnetic bead selection with the CD4+ T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications, as previously described^{35 (link)}. Anti-fibroblast microbeads (Miltenyi Biotec) were added in combination with the microbeads supplied with the kit to ensure depletion of stromal fibroblasts present in the mixed cell suspension. After two rounds of negative selection, purity of the CD4+ T cell population was higher than 90%. Isolated CD4+ T cells were activated for 24 hr with PHA and IL-2 as described for blood CD4+ T cells. 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.