Figure 3: Durability, functional phenotype, and epitope targeting of SIV insert-specific T cell responses elicited by Rh110 (pp71)-deleted RhCMV vectors in naturally RhCMV-infected RMs.

(A) Four naturally RhCMV-infected RMs were co-inoculated with 10⁷ PFU of 68–1/SIVpol-5’ and the same dose of ΔRh110/SIVrtni and flow cytometric ICS was used to follow the magnitude of the CD4⁺ and CD8⁺ T cell responses in peripheral blood to SIVpol and SIVrtn peptide mixes as described in Fig. 2. The mean + SEM of SIVpol- and SIVrtn-specific response frequencies within the memory CD4⁺ (left panel) and CD8⁺ (right panel) T cell populations are shown. (B) Boxplots compare the memory differentiation of the RhCMV-elicited CD4⁺ and CD8⁺ memory T cells in PBMCs (of the same RM shown in panel A) responding to SIVpol or SIVrtn with TNF-α and/or IFN-γ production at 630 dpi. Memory differentiation state was based on CD28 and CCR7 expression, delineating central memory (T_CM), transitional effector memory (T_TrEM), and effector memory (T_EM), as designated. The Wilcoxon rank sum test was used to pairwise compare differences between the fraction of SIVpol- and SIVrtn-specific CD4⁺ and CD8⁺ T cells within each memory subset, with P = NS for all comparisons. (C) Boxplots compare the frequency of RhCMV-elicited CD4⁺ and CD8⁺ memory T cells in PBMCs of the same RM shown in panel A responding to SIVpol or SIVrtn peptides with TNF-α, IFN-γ, IL-2, and MIP1-β production, alone and in all combinations at 630 dpi. The Wilcoxon rank sum test was used to pairwise compare differences between the fraction of SIVpol- and SIVrtn-specific CD4⁺ and CD8⁺ T cells expressing 1, 2, 3 or 4 cytokines, with P = NS for all comparisons. (D) SIVgag-specific CD8⁺ T cells in the peripheral blood of six ΔRh110/SIVgag-inoculated RMs were epitope-mapped using a flow cytometric ICS assay (CD69, TNF-α, IFN-γ readout, as described above) to detect recognition of each consecutive, overlapping 15-mer peptide comprising the SIVgag protein. Peptides resulting in specific CD8⁺ T cell responses are indicated by a box, with the color of the box designating MHC restriction as determined by blocking with the anti-pan-MHC-I mAb W6/32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP, as previously described (52,53). The blue and green arrowheads indicate the positions of previously identified MHC-II- and MHC-E-restricted SIVgag supertopes, respectively.