Profiling HPV16-Specific T-Cell Responses

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)].