2.3. Generation of virus-specific C

2.3. Generation of virus-specific CD8+ T-cell clones and cell linesThe lymphocytes isolated from the decidua basalis, decidua parietalis, and the peripheral blood were prepared for sorting as described before (Heemskerk et al., 2004). In short, cells were stained with a mixture of tetrameric complexes and antibodies directed against various cell surface molecules for 30 min at 4 °C in Iscove’s Modified Dulbecco’s Medium (IMDM) without phenol (Lonza/Biowhittaker), supplemented with 2% FBS. The antibodies used were fluorescein isothiocyanate (FITC)-conjugated and directed against CD4, CD19, CD14, CD40, CD16, and CD56 (Becton Dickinson). The cells were washed and cell sorted at 4 °C using the fluorescence-activated cell sorter Aria Sorp (Becton Dickinson). Fig. 1b shows the gating strategy used to sort tetramer-positive cells. Cells that were negative for the cell surface molecules and positive for the mixture of tetramers were sorted and cultured at 1 cell/well (clones) or 10 cells/well (cell lines). The cells were sorted into a 96-well plate containing a feeder cell mixture with irradiated allogeneic PBMCs (4000 rad), 800 ng/ml PHA, 10 IU/ml IL-2 in IMDM supplemented with glutamine (Gibco), human serum (5%) and FBS (5%). Thereafter, sorted cells were non-specifically stimulated every two weeks with freshly prepared feeder cell mixtures. CD8 and tetramer positivity was confirmed by flow cytometric analysis, as described above, when clones and/or cell lines started to expand (Fig. 1c). In addition, the mono- or polyclonality of the generated T cell clones and cell lines was confirmed by determining the TCR Vβ usage with the TCR Vβ repertoire kit (Beckman Coulter).2.4. Cytokine assaysA panel of EBV-transformed B cells (EBV LCLs) selected to cover the most frequently occurring HLA class I and II molecules were used to screen for allo-HLA cross-reactivity. In addition, mononuclear cells isolated from the umbilical cord blood of the women’s own child were used to investigate a possible fetus-specific allo-response. Supernatants were collected 18 h after co-cultures with 10,000 virus-specific T cells and 50,000 irradiated stimulator cells (EBV LCLs or umbilical cord blood cells) in a final volume of 150 μl IMDM culture medium supplemented with 10% FBS and 100 IU/ml IL-2. The concentration of IFN-γ was determined in these supernatants using a Th1/Th2 kit (Bio-Rad).2.5. Cytotoxicity assaysCell-mediated lympholysis was used to analyse the cytotoxic capacity of the generated T cell clones (effector) towards the UCB of the women’s own child (target). Maternal peripheral blood leukocytes, third party UCB with expression of HLA-A*29 and an EBV LCL with expression of HLA-A*02 (with or without the addition of the A2-EBV-GLC peptide) were used as target cell controls. The cytotoxic capacity of the effector cells was analysed in triplicate using four effector:target ratios (1:1, 5:1, 10:1 and 20:1). The target cells were labelled with 51Cr, added to the effector cells in round-bottomed 96-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO2. After incubating for 4 h, the 51Cr that was released from the target cells was measured in the supernatant of the culture using a gamma counter (PerkinElmer, Waltham, MA, USA).The percentage of specific lysis was calculated using spontaneous and maximal lysis, by incubating the target cells for 4 h in medium or 1% Triton X-100 respectively. The measured lysis was divided by the maximal lysis after correcting for the spontaneous lysis.