HIERARCHY AMONG MULTIPLE H-2(B)-RESTRICTED CYTOTOXIC T-LYMPHOCYTE EPITOPES WITHIN SIMIAN-VIRUS-40 T-ANTIGEN

Simian virus 40 large tumor (T) antigen contains three H-2D(b)-restric ted (I, II/III, and V) and one H-2K(b)-restricted (IV) cytotoxic T lym phocyte (CTL) epitopes. We demonstrate that a hierarchy exists among t hese CTL epitopes, since vigorous CTL responses against epitopes I, II /III, and IV are detected following immunization of H-2(b) mice with s yngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2D(b)-restricted epitope V was detected only following immunization of H-2(b) mice with epitope loss variant B6/K-3,1,4 cells , which have lost expression of CTL epitopes I, II/III, and IV. Limiti ng-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expa nsion and priming of epitope V-specific CTL precursors in vivo. We exa mined whether defined genetic alterations of T antigen might improve p rocessing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V i n vivo. Deletion of the H-2D(b)-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which specifies a n optimized H-2D(b) binding motif and was found to induce CTL in H-2(b ) mice, did not further reduce epitope V presentation in vitro when in serted within T antigen. Epitope V-containing T-antigen derivatives wh ich retained epitopes I and II/III or epitope IV did not induce epitop e V-specific CTL in vivo. T-antigen derivatives in which epitope V rep laced epitope I failed to induce epitope V-specific CTL. Recognition o f epitope V-H-2D(b) complexes by multiple independently derived epitop e V-specific CTL clones was rapidly and dramatically reduced by incuba tion of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CT L, suggesting that the epitope V-H-2D(b) complexes either are labile o r are present at the cell surface at reduced levels. Our results sugge st that processing and presentation of epitope V is not dramatically a ltered (reduced) by the presence of immunodominant CTL epitopes in T a ntigen and that the immunorecessive nature of epitope V is not determi ned by amino acids which flank its native location within simian virus 40 T antigen.