4.3. CMV-Specific T-Cell Response Determined by IFN-γ ELISpot

Functionality of the different T-cell fractions were analyzed by IFN-γ ELISpot assay with (target cell-dependent) and without target cells (target cell-independent). The target cell-independent assay was performed as described previously [62,63]. Briefly, PBMCs and the different T-cell fractions were isolated on day 0 and rested overnight in T-cell culture medium Roswell Park Memorial Institute medium 1640 (RPMI1640) (Lonza, Vervies, Belgium) at 1 × 10⁷ cells per ml/well with 10% heat-inactivated human AB serum (TCM) (C.C.pro, Oberdorla, Germany) in a 24 well tissue culture plate (Sarstedt, Nümbrecht, Germany) at 37 °C and 5% CO₂. On day 1, cells were resuspended with fresh medium, counted and seeded at a concentration of 2.5 × 10⁵ PBMCs as well as 5 × 10⁴ effector T cells cells/well in a 96 well plate (pre-coated anti-IFN-γ ELISpot plate; Lophius Biosciences, Regensburg, Germany) and overnight stimulation with 1 µg/mL 15-mer overlapping peptide pools (pp) of ppCMV_pp65 or ppCMV_IE1 (JPT Peptide Technologies, Berlin Germany). The target cell-dependent assay utilized target cells (CD3 negative cells), rested on day 0 in TexMACS medium (Miltenyi Biotec, Germany) and seeded at a density of 1×10⁷ cells per mL/well. Target cells were then stimulated overnight with 1 µg/mL ppCMV_pp65 or ppCMV_IE1 per 1×10⁷/mL. On day 1, the T-cell fractions (effector cells) were seeded at a concentration of 5×10⁴ T effector cells with the target cells in effector: target (E.T) ratios of 1:1 and or 2:1 in IFN-γ ELISpot plate. Untreated cells served as negative control and for positive control, cells were stimulated with 1µg/mL SEB (staphylococcus enterotoxin B) (Sigma-Aldrich, Hamburg, Germany) diluted in TCM. IFN-γ secretion was detected following 16 h of incubation at 37 °C and 5% CO₂ using biotin-conjugated antihuman IFN-γ antibodies (mAb 7-B6-1-biotin, Mabtech, Stockholm, Sweden) and streptavidin-alkaline phosphatase (Mabtech) revealed by 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT Liquid Substrate, Sigma-Aldrich, Germany). The data were acquired on an ‘AID iSpot Reader System’ (AID GmbH, Straßberg, Germany) with ‘AID ELISpot Software Version 7.0′ (https://www.aid-diagnostika.com) and spot counting was performed with ‘AID ELISpot Software Version 8.0′ (https://www.aid-diagnostika.com). All spot counts are mean values of duplicate wells and expressed as spot-forming unit per well per 100,000 CD3⁺ T cells (spwT). The cut-off for positive response was at least two times higher than the negative control. All spot counts were mean values from duplicate wells.