Effect of covalent linkage of ubiquitin on the LMP1-mediated oncogenicity and LMP1-specific T-cell responses. (A) Nude mice were injected with 1 × 10⁷ LMP1- or Ub-LMP1-expressing 3T3 cells, and tumor progression was assessed for 66 days. (B) In vivo induction of CTL responses to a model epitope fused to LMP1 following immunization with DNA expression vectors. BALB/c (H-2K^d) mice were immunized intramuscularly with DNA vectors encoding LMP1 or Ub-LMP1 fused to an H-2K^d-restricted EBV nuclear antigen 1 CTL epitope (VYGGSKTSL) at the carboxy-terminal. Splenocytes from immunized mice were stimulated with H-2K^d-restricted peptide VYGGSKTSL, and CTL activity was assessed with a standard ⁵¹Cr-release assay. The data shown are representative of 2 separate experiments. (C) Ex vivo assessment of CTL responses to a model epitope fused to LMP1 following immunization with DNA expression vectors. These responses were assessed by ELISPOT assays as described in “Materials and methods.” A minimum of 5 mice from each group were assessed for the epitope-specific T-cell reactivity. The results from panels A, B, and C are expressed as mean ± standard error (SE).