Charcoal stripped human ab serum

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.

Blood Leuko Paks for cytotoxicity experiments were obtained from the DHMC Blood Donor Program. Blood donors were anonymous, no information regarding age or hormonal status was available, and only female donors were used in this study. Peripheral blood mononuclear cells (PBMCs) were recovered by standard Ficoll density gradient centrifugation. Blood CD4+T cells were isolated from PBMCs by negative magnetic bead selection using a CD4+T cell isolation kit (Miltenyi Biotec). Following isolation, CD4+T cells were stored in liquid N₂ until use. Once thawed, the CD4+T cells were resuspended in X-Vivo 15 media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped human AB serum (Valley Biomedical, Winchester, VA) prior to cytotoxicity assays (17 (link), 23 (link)).

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.