Pvdf plate

Baboon PBMCs were prepared from whole blood by Ficoll gradient centrifugation (GE Healthcare Life Science, Paris, France). Red blood cells were then lysed. Cells were harvested and washed twice at low speed to remove platelets. PBMCs were washed with media (RPMI 1640, Penistreptomycin, Glutamine, Non-essential amino acid, Pyruvic acid-Hepes, 10% of baboon sera) and added to PVDF plates (0.45 µm, Merck Millipore), which had been precoated with an IFN-γ antibody (non-human primate IFN-γ ELISPOT kit; R&D Systems, Minneapolis) according to the manufacturer’s instructions. PBMCs were then stimulated with 40 UI tuberculin-purified protein derivative (PPD; Symbiotics Corporation, San Diego, CA, USA), IL-2 (600 UI/mL) or anti-human IL-7Rα antagonist mAb (10 µg/ml). In some conditions, CD25⁺ cells from PBMCs were depleted using a phycoerythrin (PE)-labelled anti-human CD25 (M-A251) plus anti-PE microbeads (Milteny) and a negative selection with autoMACS separator (Miltenyi Biotec). Incubation was performed overnight at 37 C°. Spots were evaluated using the ELISpot reader system (AID Ispot Spectrum) with the software version 7.0. Results were expressed as number of spots per 1 × 10⁵ PBMC.

Ten milliliters venous blood was obtained from each patient on the second day after admission. Peripheral blood mononuleated cells (PBMCs) were isolated from the blood with Lymphoprep (STEMCELL Technologies, origin). Interferon-γ (IFN-γ) ELISPOT assay was used to measure HPV E1-specific T cell responses in the freshly isolated PBMCs as mentioned previously [16 (link), 20 (link)]. Briefly, PBMCs were stimulated with overlapping peptide pool (Sigma-Aldrich, USA) representing the whole E1 protein in PVDF plates (Millipore, Bedford, MA, USA) pre-coated with 1-DIK monoclonal antibodies (Machete, Stockholm, Sweden) at 2 × 10⁵ PBMCs/well. Meanwhile, PBMCs stimulated with 20 μg/mL phytohemagglutinin (PHA) (Murex Biotech Limited, Dartford, UK) served as positive controls while unstimulated PBMCs were negative controls. After being cultured overnight, spot-forming cells (SFCs) were counted by automated ELISPOT assay reader (AID ELISPOT reader system, Autoimmune Diagnostika GmbH, Strassberg, Germany). Antigen-specific T-cell responses were considered positive when the number of SFCs from the peptide-pulsed well was greater than three times as many as those in negative control [21 (link)]. The adjusted SFCs after subtracting the average negative values were expressed as SFC/10⁶ PBMCs.

T cells obtained from healthy donors were purified using magnetic beads (Miltenyi Biotec) and then transduced with TCR genes specific for the DP4/MAGE-A3_243–258 complex^{33 (link)}. IFN-γ ELISPOT assays were performed as previously described^{32 (link),34 (link),35 (link)}. Briefly, PVDF plates (Millipore, Bedford, MA) were incubated with capture mAb (1D1K; MABTECH, Mariemont, OH) at a 1:200 ratio overnight at 4 °C. After thorough washing with 2%FCS/PBS, DP4-MAGE-A3_243–258-specific T cells were then incubated with 2 × 10⁴ of the indicated stimulator cells for 20–24 hours at 37 °C. After washing with PBS, plates were incubated with biotin-conjugated detection mAb (7-B6–1; MABTECH) at a 1:2000 ratio overnight at 4 °C. After washing with PBS, plates were incubated with HRP-conjugated Streptavidin (DAKO, Carpenteria, CA) at 1:5000, and IFN-γ spots were subsequently developed. The peptides used as controls were: MAGE-A3_243–258 (₂₄₃KKLLTQHFVQENYLEY₂₅₈) (Genway, San Diego, CA) and tetanus toxin_947–967 (₉₄₇FNNFTVSFWLRVPKVSASHLE₉₆₇) (Genway, San Diego, CA).

IFN-γ ELISPOT analysis was conducted as described previously (Kagoya et al., 2018 (link)). PVDF plates (Millipore, Bedford, MA) were coated with the capture mAb (clone 1-D1K; MABTECH, Mariemont, OH), and T cells were incubated with 2 × 10⁴ target cells per well in the presence or absence of a peptide for 20–24 hr at 37°C. The plates were subsequently washed and incubated with a biotin-conjugated detection mAb (clone 7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added, and IFN-γ spots were developed. The reaction was stopped by rinsing thoroughly with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

Antigen-specific IFN-γ release was detected by enzyme-linked immunospot (ELISpot) assay according to the manufacturers' instructions (human IFN-γ ELISpot kit: U-CyTech, Utrecht, Netherlands; mouse IFN-γ ELISpot kit: BD Biosciences). Briefly, 96-well PVDF plates (Millipore) were coated with anti-human or anti-mouse IFN-γ coating antibody overnight at 4°C. Freshly isolated cells (2.5 × 10⁵ in 200 μL culture medium) were incubated with 10 μg/mL M.tb antigen (LppZ, E6C10 fusion protein, Ag85A), tuberculin PPD (Statens Serum Institut, SSI, Copenhagen, Denmark), 5 μg/mL Concanavalin A (ConA) or 2.5 μg/mL phytohemagglutinin (PHA) (both from Sigma Aldrich) for 20 h, respectively. IFN-γ secreting cells were detected by biotin-labeled detection antibody and horseradish peroxidase (HRP) conjugated streptavidin. Coloration was developed with AEC substrate solution for 30 min at room temperature in the dark. The reaction was stopped by thoroughly rinsing the PVDF membrane with demineralized water. The plates were air-dried and the spots were counted by ELISpot Reader (BioReader Model 4000; Bio-Sys GmbH, Karben, Germany). The number of antigen-specific IFN-γ producing cells was calculated as spot-forming units (SFUs) per 2.5 × 10⁵ cells.