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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. 5.

Fig. 5

The impact of the increase in the number of immunizing doses of recombinant adenovirus (re-Ad) and recombinant vaccinia virus (re-MVA) on the induction of ANYNFTLV, a peptide derived from Trypanosoma cruzi surface antigen-specific CD8+ T cells, both the quantity and their memory phenotype. B6 mice were first primed with 1 × 104 plaque forming units (PFU) of either recombinant influenza (re-FLU) or the control FLU, and then were given a booster immunization 14 days later with either 5 × 107 PFU of re-Ad (shown as re-Ad*) or 5 × 108 PFU of re-Ad. The mice were further immunized 14 days later with either 5 × 107 PFU of re-MVA (shown as re-MVA*) or 5 × 108 PFU of re-MVA. The mice were sacrificed 12 days after the last immunization and spleens were removed. (A) 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 × 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. Data represent the mean ± S.D. of three to nine mice in each group. **, P < 0.01 determined by the Student's t-test. (B) The splenocytes were also subjected to the flow cytometory (FCM) analyses for detection of Kb/ANYNFTLV pentamer-reactive CD8+ T cells. The T-cell receptor (TCR) was stained by the phycoerythrin (PE)-conjugated Kb/ANYNFTLV pentamer, and the CD8 was stained by the PerCP-Cy5.5-conjugated anti-CD8 monoclonal antibody (mAb). The number of ANYNFTLV+, CD8+ T cells per 106 splenic lymphocytes was calculated. Data represent the mean ± S.D. of three to nine mice in each group. **, P < 0.01 determined by the Student's t-test. (C) The representative data of the FACS analyses for detectionn of Kb/ANYNFTLV pentamer-reactive or non-reactive CD8+ T cells. 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. TCM: T cells of central memory phenotype, TEM: T cells of effector memory phenotype. All the experiments were repeated at least twice for the confirmation of reproducibility.