Tetanus toxoid

To detect T‐cell responses, PBMC (1 × 10⁵) were cultured with Aspergillus fumigatus extract (Allergon, Angelholm, Sweden), tetanus‐toxoid (Statens Serum Institute, Denmark) and the MHC Class II peptide mix incorporating peptides from CMV, EBV, influenza and tetanus (CMV‐EBV‐Flu‐Tet peptide pool; JPT Peptide Technologies, Berlin, Germany). Phytohaemagglutinin (PHA; Sigma, MO, USA) was included as the positive control and PBMC alone in the absence of antigen included to assess background levels of proliferation. After 4 days 30 μL of supernatant was removed from each culture and stored at −80°C for cytokine analysis. The PBMC cultures were then supplemented with 30 μL fresh media containing 1 μCi of methyl‐³H Thymidine (³HTdR; MP Biomedicals, Australia) and harvested 16–20 h later. Incorporation of ³HTdR was measured by liquid scintillation spectroscopy as counts per minute (cpm), and mean counts per minute with standard deviation (SD) was calculated for triplicate cultures (SD < 20% for all experiments). Results are expressed as Stimulation Index (SI) which is the ratio of cpm for proliferation in presence of antigen/proliferation in PBMC alone culture and an SI > 2.5 was defined as a positive antigen‐specific response.

Panels of overlapping 30–35 mer peptides with HPV16 E2, E6, and E7 protein sequences were synthesized by solid phase peptide synthesis (SPPS) method with >95% purity (ChinaPeptides Co. Shanghai, China), with a 14 (for 30-mer) or 15 (for 35-mer) amino acid (aa) overlap. Two pools of E2 peptides (E2.1 and E2.2) consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 and two pools of E7 peptides (E6.1-E6.4 and E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively. The peptides are detailed in Table 1. Peptide quality was tested by mass spectrum and high-performance liquid chromatography (HPLC). Lyophilized peptides were dissolved in distilled water or in water with 10% DMSO for proper dissolution. Peptides were stored at −20°C with the final concentration of 1 mg/mL. Eight peptide pools were used to determine the proliferative capacity of HPV16-specific T-cells, for cytokine production analysis and detection of the HPV16-specific Foxp3+ regulatory T-cells. Memory response mix (MRM) stock solution (50×), consisting of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), Tuberculin PPD, 5 μg/mL (Statens Serum Institut), and Candida albicans, 0.015% (Greer Laboratories, Lenoir, USA) was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs [13 (link)].

PBMCs (1 × 10⁵/well) were incubated in triplets at 37 °C and 5% CO₂ in 96-well round bottom plates with the aluminum hydroxide-free and human albumin-free TBE antigen (FSME Ticovac strain Neudörfl, Pfizer, 0.6 µg/mL), the CEF MHC-I control peptide pool (Anaspec, 32 peptides, 2.5 µg/mL per peptide), a positive control peptide mix consisting of CEFT MHC-II peptide pool (JPT Peptides, 14 peptides, 0.25 µg/mL per peptide, each corresponding to a defined HLA class II restricted T-cell epitope from cytomegalovirus (CMV), Epstein–Barr virus (EBV), influenza virus or Clostridium tetani), tetanus toxoid (Statens Serum Institute, Copenhagen, Denmark, 0.125 Lf/mL) and tuberculin purified protein derivative PPD (Statens Serum Institute, Copenhagen, Denmark, 0.5 µg/mL); further, a mix consisting of phorbol myristate acetate (Sigma, 1.25 × 10⁻⁸ M) and ionomycine (Sigma, 0.1 µg/mL) or medium alone (RPMI, HyClone plus 2% Human Serum, Sigma). After 120 h cells were pulsed with methyl-[3H]thymidine (1 µCi/well) for 18 h and T-cell proliferation was quantified on a MicroBeta2 Microplate Counter (PerkinElmer, Waltham, MA, USA) as counts × 1000/min. Data was then standardized based on unstimulated controls of each patient.

Eight peptide pools of HPV16 peptides were used to determine the proliferative capacity of HPV16-specific T-cells, for cytokine polarization analysis and detection of the HPV16-specific Foxp3+ regulatory T-cells, as described previously [10 (link), 21 (link), 22 (link)]. Panels of overlapping 30-35 mer peptides with HPV16 E2, E6, and E7 protein sequences were synthesized by solid phase peptide synthesis (SPPS) method with >95 % purity (ChinaPeptides Co. Shanghai, China), with a 14 (for 30-mer) or 15 (for 35-mer) amino acid (aa) overlap. Two pools of E2 peptides (E2.1 and E2.2) consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 and two pools of E7 peptides (E6.1-E6.4 and E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively [10 (link)]. The numbers of covered amino acids of each protein are shown in Additional file 1. Memory response mix (MRM) stock solution (50×), consisting of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), Tuberculin PPD, 5 µg/mL (Statens Serum Institut), and Candida albicans, 0.015 % (Greer Laboratories, Lenoir, USA) was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs.

Eight peptide pools of HPV16 peptides were used to determine the proliferative capacity of HPV16-specific T-cells, and the cytokine polarization and frequency of HPV16-specific Foxp3+ regulatory T-cells, as described previously [15 (link), 37 (link), 39 (link)]. 30 (or 35) amino-acid-long peptides with 14- (or 15-)mer overlap covering the HPV16 E2, E6, and E7 protein sequences were synthesized by the solid-phase peptide synthesis (SPPS) method with >95% purity (ChinaPeptides Co., Shanghai, China). Two pools of E2 peptides (E2.1 and E2.2) consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 (E6.1–E6.4) and two pools of E7 peptides (E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively [15 (link)]. Memory response mix (MRM) stock solution (50×), consisting of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), tuberculin purified protein derivative (PPD), 5 μg/mL (Statens Serum Institut), and Candida albicans, 0.015% (Greer Laboratories, Lenoir, NC, USA) was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs [15 (link), 39 (link)].