Characterization of T-cell responses induced by vaccination with αDEC-205-EBNA1 and polyICLC in huNSG mice. (A) αDEC-205-EBNA1 plus polyICLC vaccination primes specific T cells in huNSG mice. HuNSG mice were vaccinated with 5 μg of IgG-EBNA1 or αDEC-205-EBNA1 with the use of 50 μg of polyICLC as adjuvant and boosted with the same dose of antibodies and adjuvant 4 weeks later. The mice were euthanized 6 to 8 weeks after the boost. T-cell clones were generated by limiting dilution cloning of specific T cells after an IFN-γ capture assay after restimulation with 5μM EBNA1 peptide library for 12 hours. IFN-γ secretion was analyzed by ELISA in the supernatant. (B) The specificity of 3 expanded T-cell clones (c15, c16, c21) are shown after restimulation with 5 different peptide subpools of EBNA1 by measuring IFN-γ secreted into the supernatant. Data are represented as mean ± SD from duplicate or triplicate wells of IFN-γ ELISA. (C) The EBNA1 specific T-cell clone c16 recognizes autologous LCLs. Autologous LCLs (Auto LCL) or allogeneic LCLs (Allo LCL), unmanipulated or loaded for 1 hour with 5μM EBNA1 peptides, were incubated with the expanded EBNA1-specific T-cell clone c16 (E:T=1:2), T-cell activity was determined after 18 hours by measuring released IFN-γ by ELISA. (D) Same as in panel C but analyzing T-cell function by CD107a staining after 6 hours of coculture. (E) The T-cell response of clone c16 is MHC class II-restricted. T-cell clone c16 was exposed to the autologous LCLs in the absence or presence of the indicated HLA blocking antibodies and IFN-γ was measured in the supernatants by ELISA. One representative experiment of 2 is shown. Statistical analysis was performed by Mann-Whitney U tests, and data are represented as mean ± SD.