Positive Selection of CD8-Positive

Positive Selection of CD8-Positive LymphocytesFollowing overnight incubation, CD8-positive lymphocytes were positively selected from the nonadherent decidual cells. Decidual cells were first incubated with anti-CD8 murine monoclonal antibody (clone DK25; DAKO, Cambridge, UK), followed by anti-mouse immunoglobulin G-coated magnetic microbeads (MACS; Miltenyi Biotech Ltd, Surrey, UK); CD8+ cells were positively selected using a MidiMACS magnetic separation system following the manufacturer's protocol. Purity was routinely confirmed by either flow cytometry or immunohistochemistry on cell smears. For flow cytometry, purified cells were treated with CD8 fluorescein isothyocyanate/CD56 phycoerythrin/CD3 peridinin chlorophyll-A protein-cytochrome 5.5 combination antibody (BD Biosciences, Aalst, Belgium). For immunohistochemistry, cell smears were labeled using a modified avidin-biotin complex (ABC) method using the Vectastain Elite kit (Vector Laboratories, Peterborough, UK). Briefly, the smears of positively selected cells (already incubated during selection with anti-CD8 antibody) were incubated sequentially with appropriately diluted biotinylated horse anti-mouse immunoglobulins (Vector Laboratories; 30 min) and the Vectastain ABC-peroxidase reagent (Vector Laboratories; 30 min). The reaction was developed using NovaRed (Vector Laboratories) to give a red reaction product. Smears were lightly counterstained with Mayer hematoxylin, blued in Scott tap water, dehydrated, cleared, and mounted in DPX (BDH).Cytotoxicity AssaysDecidual CD8+ effector cell cytotoxic function was assayed either in a standard 4-h chromium−51 (51-Cr) release assay against the human erythroleukemia cell line K562 target cells to assay NK cell-sensitive cytotoxicity, as described previously [22], or in a CD3 antibody redirected chromium release assay against the murine mastocytoma Fcγ cell line P815 target cells that require T-cell receptor signal transduction to mediate cytotoxicity [5]. For the redirected chromium release assay, decidual CD8+ T cells were assessed at 7 wks GA (n = 1), 8 wks GA (n = 2), 9 wks GA (n = 1), 10 wks GA (n = 3), 12 wks GA (n = 1), 13 wks GA (n = 2), and 14 wks GA (n = 1); and for the standard chromium release assay at 8 wks GA (n = 2) and 13 wks GA (n = 1). The K562 and P815 cell lines were a kind gift from Dr. Colin Brooks, University of Newcastle-upon-Tyne. 51-Cr-labeled P815 cells were preincubated in 10 μl/ml CD3 murine monoclonal antibody (clone UCHT1, DAKO) for 30 min at 37°C in 5% CO2. Labeled target cells (1x104 in 100 μl complete medium) were incubated with a range of doubling dilutions of decidual CD8+ effector lymphocytes to give final effector:target (E:T) ratios of between 60:1 and 1:1 in triplicate in a final volume of 200 μl in round-bottomed plates. The highest E:T ratio was determined by the yield of decidual effectors. The plates were centrifuged at 200 g for 10 sec, incubated at 37°C in 5% CO2 for 6 h, centrifuged at 200 g for a further 10 sec, and then 100 μl of each supernatant was harvested and counted in a gamma counter to determine isotope release. Minimum and maximum 51Cr release was determined in 10% FCS and 30% detergent solution (Hospec General Purpose Neutral liquid detergent; Youngs Detergents, Cheshire, UK), respectively. Specific lysis was determined as described previously [22]. The % Specific Chromium Release (%SCR) at each E:T ratio was subjected to linear regression analysis (Microsoft Excel; Microsoft Corporation, Redmond, WA), and the mean %SCR (± SEM) at each E:T ratio for all the samples was then calculated. In order to allow comparison between the CD3 antibody redirected and the standard chromium release assays, the results are also presented as the % Specific Chromium Release at E:T ratio of 32:1 (%SCR32).Induction of Cytokine Production by CD8+ Decidual LymphocytesPositively selected CD8+ decidual lymphocytes were resuspended in complete medium (1x106 cells/ml), and 100 μl of cell suspension was dispensed into wells of round-bottomed 96-well plates. An equal volume of phytohemagglutinin-P (PHA-P; 10 μg/ml) (Sigma-Aldrich) was added to stimulate CD8+ decidual lymphocytes. Plates were incubated for 24 and 48 h. After this period, supernatants were collected, spun at maximum speed in a microcentrifuge (Eppendorf) for 2 min, and cell-free supernatants were stored at −70°C.