Figure 3.

Prime boost vaccination with rOVA-3 elicits B8R_70–78 and D1R_808–817-reactive CD8⁺ T cells. (A) Diagram showing OVA (rOVA-3) construct in which the original cryptic, OT-I and OT-II epitopes were replaced with C4R_70–78, B8R_70–78 and D1R_808—817 epitopes, respectively. A six-histidine tag at the C-terminus of rOVA-3 facilitated purification after expression in E. coli. (B) Mice were primed with rOVA-3 + αGC, boosted twice with the same vaccine, and CD8⁺ T cells isolated and purified on days shown. (C&D) Intravital staining was performed as in Fig. 1, and B8R_70–78/B0702 and D1R_808—817/B0702 tetramer-reactive cells were purified by FACS using the gating strategy shown for splenic (C) and pulmonary (D) CD8⁺ T cells. Purified pB0702 tetramer-reactive cells were used for downstream transcriptomic studies (n = 12 mice; each replicate consisted of cells pooled from 3 mice).