Chemokine-scFv fusion vaccines deliver Id to APCs for stimulation of CD4⁺ T cells in vitro as well as in vivo. (A) In vitro T-cell proliferation. Irradiated (8Gy) BALB/c splenocytes were incubated with titrated amounts of different purified chemokine-scFv fusion proteins (shown in nanomoles of scFv) and polarized Id(λ2³¹⁵)-specific Th2 cells from TCR-transgenic mice. Proliferation of T cells was measured 3 days later by incorporation of ³[H] dTdR. (B) In vivo Id priming of APCs. BALB/c mice were injected in the quadriceps muscles with 50 μg of indicated plasmids, followed by electroporation. Eight days later, draining (lumbar and sacral) and nondraining LN cells were isolated, irradiated, and tested for their ability to stimulate Id(λ2³¹⁵)-specific 7A10B2 CD4⁺ T cells in a standard 3-day proliferation assay. (C) In vivo T-cell activation. Lymph node cells from vaccinated (refer to B) or naive TCR-transgenic SCID mice were isolated on day 10 and gated clonotype⁺ CD4⁺ T cells were analyzed for CD69 expression by flow cytometry. (D) In vivo T-cell proliferation. Vaccinated (refer to B) or naive BALB/c mice were reconstituted with LN and spleen cells from TCR-transgenic mice on day 7 and injected with 2 mg BrdU intraperitoneally on day 10. On day 14, gated Id-specific CD4⁺ T cells in LNs were analyzed for BrdU expression by flow cytometry. Error bars in panels A and B indicate means plus or minus SEM.