Figure 2. Tetramer responses.

The gating strategy for T cell analysis by flow cytometry is shown in panel (a). First, lymphocytes were selected by forward and side-scatter. Next, singlets were selected, then dead cells that were ViVid positive were excluded. Next, we selected CD8+CD3+ cells, the excluded CD4+ cells before gating on tetramer-specific responses. K^dM2_82–90–tetramers were conjugated with PE, and D^bM_187–195–tetramers were conjugated with APC allowing them to be analyzed together in mice with both H-2^d and H-2^b alleles. We first evaluated BALB/c (H-2^d) mice 4 weeks after immunization with 5×10⁷ IU of HPV16-M/M2 IVag or IM. Secondary immunizations of 5×10⁷ pfu HPV45-M/M2 were given at week 4 by the same route. Negative control mice received 5×10⁷ IU HPV-luciferase IVag. All mice were treated with Depo-Provera (4 days) and N-9 (5 hours) prior to IVag immunization. RSV IN challenge occurred at week 8. On days 4 and 7 following challenge with 10⁷ pfu of A2 strain of RSV, mice were euthanized; lungs were harvested, and stained for K^dM2_82–90 tetramer-specific CD8+ T cells. Day 4 (light grey) and 7 (black) post-challenge K^dM2_82–90 tetramer-specific responses were higher in the immunized mice indicating significant priming. ** p≤0.001; *** p≤0.0001 by one-way ANOVA and T-test compared to empty vector-immunized control. N = five mice per group.