Identification of novel subdominant epitopes on the carcinoembryonic antigen recognized by CD4+ T cells of lung cancer patients

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4(+) T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4(+) T cells from the patients. We had previously identified CEA(177-189/355-367) as an immunodominant epitope recognized by CD4(+) T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA(99-111), CEA(425-437), CEA(568-582), and CEA(666-678)), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4(+) T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4(+) T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-gamma-producing CD4(+) T cells in two patients. Our data indicate that CD4(+) immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.