Figure 1.

Restriction of transgene expression to nonhematopoietic lineages has a minor influence on CD8⁺ T-cell priming. (a) The mOVA-HY construct comprises the leader peptide from the H-2K^b gene (SS), the full-length OVA cDNA, the H-2D^b transmembrane sequence (TM) and, fused at the C-terminal, the DBY₆₀₈ and UTY₂₄₆ epitopes encompassed by 4–15 amino acids of their original protein sequence to ensure normal processing. In the mOVA-HY-miR construct, an additional sequence comprising four repeats of the miR142.3p target sequence were inserted in the 3′ untranslated region. (b) Rag2^−/− female mice were injected i.m. at day 0 with rAAV1-mOVA-HY or rAAV1-mOVA-HYmiR. On day 3, mice were sacrificed and 2 × 10⁵ cells from draining or nondraining lymph nodes were cocultured with 4.10⁴ OT-1 cells. After 24 hours, CD69 activation was tested by flow cytometry. One representative experiment out of three is shown. (c–d) C57Bl/6 female mice were injected i.m. at day 0 with rAAV1-mOVA-HY (rAAV-WT) or rAAV1-mOVA-HYmir (rAAV-miR). (c) On day 14, 28, or 35 mice were sacrificed and their splenocytes were tested by IFN-γ ELISPOT assay against the MHC I–restricted OVA₂₅₇ or UTY₂₄₆ peptides. Data represent at least six to nine mice per group and per time point pooled from two independent experiments. (d) On day 14, tibialis anterior muscles from sacrificed mice were frozen and 7 µm thick transversal cryosections were stained with hematoxilin and eosin. Field size is 720 × 720 micron. One representative muscle out of three is shown.