Isolation of Purified CD8+ T Cells

Following removal of dead cells from the filter pass through suspension of stromal cells, CD8+T cells were isolated using negative magnetic bead selection with the CD8+T cell isolation kit (Miltenyi Biotec) following instructions with minor modifications. This negative selection protocol delivers untouched CD3+CD8+T cells. Additionally, 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 as described before^{9 (link),11 (link)}. After two rounds of negative selection, purity of the CD8+T cell population was higher than 90%, with approximately 2% contamination with non-immune cells, 2% CD3- cells and 1–2% contamination with CD4+T cells^{11 (link)}. The average number of total CD8+T cells recovered per gram of tissue was 2 × 10⁵. Following isolation, purified CD8+T cells were resuspended in immune cell media consisting of X-VIVO 15 Media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped human AB serum (Valley Biomedical, Winchester, VA) and co-cultured with E₂ and/or P for 48 h or TGFβ (10 ng/ml, PeproTech Inc.)^{11 (link),49 (link)} for 2 h prior to cytotoxicity assays.