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. Author manuscript; available in PMC: 2011 Nov 1.

Published in final edited form as: Int J Parasitol. 2010 Jul 8;40(13):1549–1561. doi: 10.1016/j.ijpara.2010.05.011

Fig. 3 — Characterization of recombinant influenza virus (re-FLU) for the induction of antigen-specific CD8⁺ T cells. (A) B6 mice were immunized with either re-FLU or control FLU at four different immunizing plaque forming unit (PFU) doses. They were sacrificed 12 days (Experiment 1) or 14 days (Experiment 2) after the immunization, and their spleens were removed. The freshly-isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV, a peptide derived from Trypansoma cruzi surface antigen, peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells × 10⁶ was counted 22 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. (B) B6 mice were first primed with 1 × 10⁴ PFU of re-FLU, 5 × 10⁷ PFU of recombinant adenovirus (re-Ad) or 5 × 10⁷ PFU of recombinant vaccinia virus (re-MVA). The same immunization doses of each virus were given to the mice as booster immunizations 14 days later. The mice were sacrificed 12 days after the last immunization and their spleens were removed. The freshly-isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± S.D. of three mice in each group. **, P < 0.01 determined by the Student's t-test. (C, D) The representative data of the FACS analyses for detection of K^b/ANYNFTLV pentamer-reactive or non-reactive CD8⁺ T cells. Immune splenocytes described in (B) were subjected to the flow cytometory analyses for the detection of K^b/ANYNFTLV pentamer-reactive CD8⁺ T cells. The T-cell receptor (TCR) was stained by the phycoerythrin (PE)-conjugated K^b/ANYNFTLV pentamer, and the CD8 was stained by the PerCP-Cy5.5-conjugated anti-CD8 monoclonal antibody (mAb). The ANYNFTLV⁺, CD8⁺ T cells or ANYNFTLV^-, CD8⁺ T cells were stained with the APC-conjugated anti-CD44 mAb and the FITC-conjugated anti-CD62L mAb. T_CM: T cells of central memory phenotype, T_EM: T cells of effector memory phenotype. The use of control Ad, control MVA and control FLU which do not express the epitope did not induce the ANYNFTLV-specific immune responses (data not shown). All of the experiments were repeated at least twice for the confirmation of reproducibility.

Fig. 3 — Characterization of recombinant influenza virus (re-FLU) for the induction of antigen-specific CD8⁺ T cells. (A) B6 mice were immunized with either re-FLU or control FLU at four different immunizing plaque forming unit (PFU) doses. They were sacrificed 12 days (Experiment 1) or 14 days (Experiment 2) after the immunization, and their spleens were removed. The freshly-isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV, a peptide derived from Trypansoma cruzi surface antigen, peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells × 10⁶ was counted 22 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. (B) B6 mice were first primed with 1 × 10⁴ PFU of re-FLU, 5 × 10⁷ PFU of recombinant adenovirus (re-Ad) or 5 × 10⁷ PFU of recombinant vaccinia virus (re-MVA). The same immunization doses of each virus were given to the mice as booster immunizations 14 days later. The mice were sacrificed 12 days after the last immunization and their spleens were removed. The freshly-isolated splenocytes were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± S.D. of three mice in each group. **, P < 0.01 determined by the Student's t-test. (C, D) The representative data of the FACS analyses for detection of K^b/ANYNFTLV pentamer-reactive or non-reactive CD8⁺ T cells. Immune splenocytes described in (B) were subjected to the flow cytometory analyses for the detection of K^b/ANYNFTLV pentamer-reactive CD8⁺ T cells. The T-cell receptor (TCR) was stained by the phycoerythrin (PE)-conjugated K^b/ANYNFTLV pentamer, and the CD8 was stained by the PerCP-Cy5.5-conjugated anti-CD8 monoclonal antibody (mAb). The ANYNFTLV⁺, CD8⁺ T cells or ANYNFTLV^-, CD8⁺ T cells were stained with the APC-conjugated anti-CD44 mAb and the FITC-conjugated anti-CD62L mAb. T_CM: T cells of central memory phenotype, T_EM: T cells of effector memory phenotype. The use of control Ad, control MVA and control FLU which do not express the epitope did not induce the ANYNFTLV-specific immune responses (data not shown). All of the experiments were repeated at least twice for the confirmation of reproducibility.