IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice

Intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) elicited immunity against transplanted syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells, but tumors still appeared in all mice. In wild-type mice, coinjection of an IL-12 plasmid markedly enhanced anti-CEA humoral, T-helper-1 and cytotoxic T lymphocyte (CTL) responses, and resistance to a CEA/LLC tumor challenge such that 80% of mice remained tumor free. Injection of the IL- 12 plasmid alone was not protective. To analyze immune requirements, we immunized gene knockout (KO) mice of C57BL/6 background, deficient in either CD3, CD4, CD8, interferon gamma (IFNgamma), perforin or Fas ligand (FasL). Only CD3+ mice expressing both CD4 and CD8, which appear equally important, as well as IFNgamma and perforin, could fully resist a tumor challenge. IL-12 stimulated CTL activity, which was strictly CD3/CD8/perforin-dependent. FasL-KO mice had normal CTL activity and tumor resistance, indicating that only the perforin lytic pathway was involved. CD4-KO and IFNgamma-KO mice still generated CTLs. CEA-stimulated IFNgamma production occurred in both CD4- or CD8-KO mice and in both cases was augmented by IL-12. In IFNgamma-KO mice, IL-12 still enhanced anti-CEA antibody production but only moderately restored impaired DTH and tumor resistance. We conclude that the immune requirements for tumor rejection are stringent, involving multiple mechanisms which are all enhanced by IL-12.