Figure 3.

Phenotypic characterization of vaccine-induced Nef RL10-specific CTLs during the vaccination phase. Vaccinee PBMCs were stained with fluorophore-conjugated Nef RL10-Mamu-B*08 tetramers and fluorophore-conjugated mAbs to assess the following CTL phenotypic characteristics: (A) frequencies of granzyme B⁺ CTLs, (B) frequencies of Ki-67⁺ CTLs, and (C) frequencies of terminally-differentiated effector memory (T_EM2) CTLs, defined as CD28^– CCR7^– CTLs. PBMCs from the following timepoints were analyzed: two weeks post-rAd5 vaccination (“rAd5”), two weeks post-rVSV vaccination (“rVSV”), two weeks post-rRRV vaccination (“rRRV”), at the time of the first SIVmac239 challenge (“TOC 1”), and retrospectively at the time of the infecting challenge for a given animal (“SIV+”). Plots depict frequencies of Nef RL10-Mamu-B*08 tetramer⁺ CD8⁺ T cells (defined as live tetramer⁺ CD3⁺ CD8⁺ CD4^– CD14^– CD20^– CD159a^– lymphocytes) meeting the staining criteria for each phenotypic characteristic. Statistical significance testing was performed to assess differences in Nef RL10-specific CTL phenotypic characteristics across all timepoints (Friedman test) and to determine whether these phenotypic characteristics changed during the challenge phase (Wilcoxon test, comparing the “TOC 1” and “SIV+” timepoints). Friedman test P-values were < 0.0001, < 0.0001, and 0.02073 for panels (A-C) respectively. Wilcoxon test results are shown in each panel: *P < 0.05, **P < 0.01.