Pre coated elispot plates

In vitro expansion using whole RIT and T-cell activation detection using IL-2 ELISpot were performed as previously described [30 (link)]. Briefly, PBMC from naïve donors were stimulated with 5μg/ml of SS1P, LMB-T14 or LMB-T20 in separate plates. The cells were supplemented with recombinant human IL-2 every 4 days (Millipore). On day 14, the cells were harvested and washed. They were brought to a concentration of 2×10⁶cells/ml and 50 μl were plated in pre-coated ELISpot plates (Mabtech). The enriched cells were then restimulated with peptide pools; cells that were expanded with SS1P were restimulated with 22 peptide pools spanning the sequence of WT PE38. Cells that were expanded using LMB-T14 or T20 were restimulated with 15 peptide pools spanning the sequence of the deimmunized RIT. Peptide pools that had a positive responses as defined [31 (link)] were fine screened to identify the individual immunogenic peptides by testing individual peptides from the pool.

Human IFN-γ secretion was measured by stimulation of 2x10⁵ PBMCs in RPMI 1640 supplemented with 10% FBS using precoated ELISpot plates (Mabtech). Processed plates were analyzed by Zellnet Consulting. Samples were run in triplicate wells.

Enzyme-linked immunospot assay (ELISPOT) was performed according to the manufacturer’s instructions (MabTech, Sweden). Briefly, 15-mer peptides of VZV–gE were prepared: each peptide had an 11-amino acid overlap to cover the entire sequence of the gE protein. For the assay, an optimal number (5 × 10⁵ cells per well) of single cells were inoculated onto precoated ELISPOT plates (MabTech). Cells were then incubated with pooled VZV–gE peptides and stimulated for 20 h. Spots were developed according to the manufacturer’s instructions. Spots were scanned and quantified using a CTL-ImmunoSpot S5 reader. The number of spot-forming cells (SFC) was calculated by subtracting the mean number of PBS–stimulated wells. A mix of phorbol 12-myristate 13-acetate (PM, 20 ng/mL) and ionomycin (Sigma-Aldrich, 1 μg/mL) served as the positive control, with PBS used as the negative control.