A control H2-Kb tetramer constructed using the HSV1-gB498C505 peptide specifically stained the 2D5 CTL clone (panel d). (1 in 67,000) and epitope V-specific CTL were undetectable ATF1 (41). Although epitope V-specific CTL are not detected following immunization with full-length SV40 Tag, immunization with syngeneic cells carrying inactivating mutations or deletions in Tag epitopes I, II/III, and IV leads to the induction of epitope V-specific CTL (41, 50). Accordingly, epitope V has been characterized as immunorecessive. Additional strategies which enhance the immunogenicity of epitope V include immunization with rVVs which express epitope V as a minigene linked to a secretory signal sequence (ES) or Flufenamic acid in which the epitope V sequence is inserted into a nonimmunogenic murine self protein, dihydrofolate reductase (26). Precise mechanisms which control the immunorecessive nature of epitope V from within the Tag are not known, although the epitope V peptide forms unstable complexes with H2-Db molecules, may be degraded rapidly during antigen processing, and is located between flanking sequences in the Tag, which may limit antigen processing (26, 40). In this study, we analyzed the hierarchy of Tag-specific CD8+ T lymphocyte responses by using direct methods in light of recent studies which have suggested that traditional cytotoxicity-based analyses, which require in vitro restimulation, can substantially underestimate the frequencies Flufenamic acid of pathogen-specific T lymphocytes (14, 33, 39). Therefore, we investigated the hierarchy, persistence, and T-cell receptor (TCR) diversity of Tag epitope-specific CD8+ T lymphocytes by using major histocompatibility complex (MHC) class I tetramers or intracellular gamma interferon (IFN-) accumulation following immunization of C57BL/6 mice with infectious SV40, Tag-expressing syngeneic cells, or rVVs expressing full-length Tag or the individual Tag epitopes. The rationale for the use of three different vehicles to deliver Tag to the immune system was that potentially different modes of antigen presentation of Tag might result in quantitative differences in the Tag epitope-specific CD8+ T-lymphocyte responses. Tag delivered by transformed cells sensitizes CD8+ T lymphocytes by cross-priming due to a lack of costimulatory molecules (29), whereas contamination with live SV40 or rVVs may target a variety of cells including antigen presenting cells (24, 37, 38). The results of direct ex vivo analysis of Tag epitope-specific CD8+ T lymphocytes support the establishment of a hierarchical relationship in vivo in which IV I II/III V. Epitope IV-specific CD8+ T lymphocytes dominated following immunization of C57BL/6 mice with Tag-transformed Flufenamic acid cells, SV40, or rVV-941T, which express full-length Tag. Epitope V-specific CD8+ T lymphocytes remained undetectable following immunization with full-length Tag but were recruited to dramatically increased levels in vivo Flufenamic acid by immunization and boosting with transformed cells which express the epitope I-, II/III-, and IV-deficient Tag derivative. In addition, TCR repertoire analysis of Tag epitope-specific CD8+ T cells indicated a lack of correlation between the amount of TCR diversity observed and the hierarchy of the CD8+ T-cell response. Flufenamic acid MATERIALS AND METHODS Animals. Male or female C57BL/6 (cell lines were cultured as described previously (26) and harvested by trypsinization prior to use in immunization or cytotoxicity assays. For studies involving direct ex vivo staining of Tag-specific splenic CD8+ cells, groups of three to five C57BL/6 mice were routinely immunized with 4 107 to 5 107 Tag-transformed cells by the intraperitoneal route. Subsequent boosting with Tag-transformed cells was done by the same method at the indicated occasions. C57BL/6 mice were immunized in the hind footpads with 3 106 PFU of SV40 strain VA45-54 in 50 l of phosphate-buffered saline (PBS) as described previously (43). Immunization of C57BL/6 mice with rVVs which express SV40 Tag or individual Tag CTL epitopes was performed as described previously.