INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Immunity to the large tumor antigen (Tag) of simian virus 40 (SV40) in C57BL/6 mice is characterized by the development of CD8⁺ T lymphocytes restricted by both H2-D^b and H2-K^b and directed toward multiple epitopes within the Tag (54). Four distinct H2^b-restricted CD8⁺ T-lymphocyte epitopes, epitopes I, II/III, IV, and V, have been identified and precisely mapped within SV40 Tag. Epitope IV, Tag residues 404 to 411, is H2-K^b restricted (42, 52). Epitopes I (residues 206 to 215), II/III (residues 223 to 231), and V (residues 489 to 497) are restricted by H2-D^b (23, 34, 50, 52). An immunological hierarchy has been demonstrated among these four epitopes within Tag. Immunization of C57BL/6 mice with SV40, SV40 Tag-transformed cells, or a recombinant vaccinia virus (rVV) which encodes the full-length Tag leads to the induction of cytotoxic T lymphocytes (CTL) specific for epitopes I, II/III, and IV (26, 41, 51). Frequency estimates from limiting-dilution analysis of splenic lymphocytes obtained 9 days after immunization with SV40 Tag-transformed cells revealed that epitope IV-specific CTL represent 1 in 14,000 splenocytes while epitope I and II/III-specific CTL were less abundant (1 in 67,000) and epitope V-specific CTL were undetectable (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 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-D^b 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 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 infection 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 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 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.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. Male or female C57BL/6 (H-2^b) mice (4 to 6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, Maine) and routinely used between the ages of 7 and 12 weeks. All mice were maintained in the animal facility at Pennsylvania State University College of Medicine (Hershey, Pa.).

Cell lines and viruses. SV40-transformed B6/WT-19 cells have been described previously (45). B6/T116A1 cells were generated by calcium phosphate-mediated transfection of mouse embryo fibroblasts with plasmid pSLM361-11 as described previously (41). Plasmid pSLM361-11 was generated by site-directed mutagenesis of plasmid pLM247 (41) using the Altered Sites mutagenesis procedure (Promega Corp., Madison, Wis.). B6/T116A1 cells express a Tag derivative in which epitopes I (residues 207 to 215) and II/III (residues 223 to 231) are deleted and epitope IV is inactivated by alanine substitution of residues 406, 408, and 411. B6/T122B1 cells express a Tag derivative in which all four determinants (I, II/III, IV, and V) have been inactivated by virtue of alanine substitutions to critical MHC class I anchor residues (N210A, N227A, F408A, and N493A). Plasmid pLMTS364-1 was used to establish the B6/T122B1 cells and was derived from pSelect-ESV-1 by site-directed mutagenesis as described (41). B6/T122B1 cells are not specifically lysed by SV40 Tag-specific CTL clones or Tag-specific bulk CTL (data not shown). RMA cells have been described previously (36).

CTL clones. The SV40 Tag-specific CTL clones K-11, K-19, Y-4, and H-1 have been described previously (15, 41, 50, 51). Conditions for culture and use of these CTL clones have been described previously (41). CTL clones ESV-F8 and ESV-F9 were derived by limiting-dilution cloning from an epitope V-specific CTL line. Epitope V-specific CTL lines were established by repeated subculture of splenocytes obtained from C57BL/6 mice immunized with the epitope V-minigene expressing rVV strain rVV-ES-V (26) in the presence of recombinant human interleukin-2 (Amgen) and irradiated B6/K-3,1,4 cells essentially as described previously (41, 50). Epitope IV-specific CTL clones 15Bb/w-C7 and 15Bb/w-C18 were derived by limiting-dilution cloning from an epitope IV-specific CTL line generated by immunization of C57BL/6 mice with B6/T15Bb cells which express a Tag derivative from which epitopes I, II/III, and V have been deleted (41). The epitope IV-specific CTL line was established by repeated subculture in the presence of recombinant human interleukin-2 and irradiated B6/WT-19 cells essentially as described previously (51).

Induction of SV40 Tag-specific CTL and generation of bulk CTL. Tag-transformed H2^b 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 times.

Cytotoxicity assays. Cytotoxicity assays were used to verify the presence of SV40 Tag-specific CTL in splenic or lymph node-derived cell populations following in vitro restimulation with gamma-irradiated Tag-transformed cells. Lysis of Tag-transformed or peptide-pulsed target cells by C57BL/6-derived, secondary in vitro-restimulated lymphocytes was measured by using a standard ⁵¹Cr release assay as previously described (26).

Production of MHC class I tetramers. Synthetic peptides used for tetramer construction were synthesized in the Macromolecular Core Facility of the Pennsylvania State University College of Medicine as described previously (41). Peptides were used for tetramer construction without further purification; these were Tag epitope I (SAINNYAQKL), epitope II/III (SKGVNKEYL [C223S]), epitope IV (VVYDFLKL [C411L]), epitope V (QGINNLDNL), DbN5 peptide (SMIKNLEYM), and herpes simplex virus type 1 (HSV-1) gB peptide (SSIEFARL) (5, 7, 23, 27, 34, 41, 42, 50, 51). Underlined residues indicate substitutions which were incorporated to avoid potential problems with cysteine oxidation. Substituted peptides were found to sensitize targets for lysis by appropriate SV40 Tag-specific CTL clones and bulk Tag-specific CTL either as well as or better than did peptides corresponding to the wild-type Tag epitopes (data not shown).

FACS analysis of tetramer-stained lymphocytes. Splenic lymphocytes or bulk CTL were washed twice in fluorescence-activated cell sorter (FACS) buffer (PBS supplemented with 2% [vol/vol] fetal bovine serum and 0.1% [wt/vol] sodium azide), resuspended at 2 × 10⁷ cells per ml in FACS buffer, and incubated in the presence of rat anti-mouse CD16/CD32 (33 µg/ml [FC Block; Pharmingen]) and unconjugated streptavidin (33 µg/ml) (Molecular Probes) for 1 h on ice. Cells were diluted to 14 ml with FACS buffer, pelleted, and resuspended to 2 × 10⁷ cells per ml in ice-cold FACS buffer. Aliquots (100 µl) were added to ice-cold microcentrifuge tubes containing 2 µl each of tetramers and antibodies, at which time all the components were combined and mixed briefly by vortexing. Suspensions were incubated on ice for at least 1 h with intermittent mixing. Staining suspensions were diluted into 4 ml of FACS buffer, pelleted, washed with 4 ml of FACS buffer, pelleted, and resuspended in 0.1 ml of PBS containing 2% (wt/vol) paraformaldehyde. Samples were analyzed promptly on a Becton Dickson FACScan instrument using CELLQuest software. Routinely, 1 × 10⁵ to 2 × 10⁵ cells were analyzed for tetramer-staining analysis of ex vivo splenic populations while 1 × 10⁴ to 5 × 10⁴ cells were collected for analysis of bulk-cultured CTL or CTL clones. In instances where staining with unrelated control tetramers is not shown, background staining was accounted for by subtracting the number of CD8⁺ cells which stained with a control tetramer from the number of CD8⁺ cells which stained specifically, as indicated in the figure legends.

Intracellular cytokine assay. For staining of intracellular IFN-, spleen cells were harvested from mice at the indicated times postimmunization. RBC-depleted spleen cell suspensions were prepared, and 5 × 10⁶ spleen cells were incubated for 6 h at 37°C under 5% CO₂ with 1 µM indicated synthetic peptides representing Tag or control epitopes and 1 µg of brefeldin A (Sigma) per ml in 2 ml of complete RPMI 1640 containing 10% fetal bovine serum per well of a 24-well plate. Cells were stained for intracellular IFN- using the Cytofix/Cytoperm Kit (Pharmingen) as specified by the manufacturer. Briefly, stimulated cells were washed twice, Fc receptors were blocked by incubation with rat anti-mouse CD16/CD32 (Fc Block; Pharmingen) for 20 min, and the cells were stained with PE-labeled rat anti-mouse CD8 (Pharmingen) for 30 min. After fixation and permeabilization for 20 min, the cells were stained with fluorescein isothiocyanate-labeled rat anti-mouse IFN- (Pharmingen) or an isotype control antibody for 30 min and then analyzed by flow cytometry as described above. The percentage of CD8⁺ cells which express intracellular IFN- was calculated by subtracting the percentage of cells which stained nonspecifically with the isotype control antibody from the percentage which stained specifically for IFN- following stimulation with the indicated synthetic peptide. In some instances, as indicated in the figure legends, the number of CD8⁺ T cells which stained for IFN- in the presence of control peptide was additionally subtracted to determine the number of CD8⁺ T cells which stained specifically for IFN-.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of SV40 Tag-specific MHC class I tetramers. To directly enumerate and characterize SV40 Tag-specific CD8⁺ T lymphocytes, MHC class I tetramers were constructed using synthetic peptides corresponding to four SV40 Tag CTL epitopes (I, II/III, V, and IV). The specificities of the tetramers were verified by testing for reactivity against SV40 Tag-specific CTL clones (Fig. 1). Due to the presence of a COOH-terminal cysteine residue in the 8-mer epitope IV peptide (VVYDFLKC), the epitope IV tetramer reagent (Kb/IV Tet) was constructed using a C411L-substituted synthetic epitope IV peptide (VVYDFLKL). The C411L-substituted epitope IV peptide was recognized more efficiently than the synthetic wild-type epitope IV peptide by the epitope IV-specific CTL clone Y-4 or by epitope IV-reactive bulk CTL (data not shown). Kb/IV Tet specifically stained the CTL clone Y-4 (Fig. 1A, panel a) but did not stain CTL clone 2D5, which is H2-K^b restricted and specific for residues 498 to 505 of HSV-1 gB (7). A control H2-K^b tetramer constructed using the HSV1-gB498-505 peptide specifically stained the 2D5 CTL clone (panel d).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Specificity of MHC class I tetramers constructed using SV40 Tag epitopes or control peptides demonstrated by staining of SV40 Tag-specific CTL clones. (A) Costaining of H2-Kb-restricted CTL clones Y-4 (Tag epitope IV specific) and 2D5 (HSV-1 gB specific) with anti-CD8 and Kb/IV Tet or Kb/gB Tet. (B) SV40 Tag-specific H2-Db-restricted CTL clones K-11 (Tag epitope I specific), K-19 (Tag epitope II/III specific), and H-1 (Tag epitope V specific) were stained with anti-CD8 and MHC class I tetramers Db/I Tet, Db/II/III Tet, Db/V Tet, or an unrelated Db tetramer, Db/DbN5 Tet.

Quantitation of SV40 Tag-specific CD8⁺ lymphocytes following immunization with syngeneic SV40 Tag-transformed cells. To quantitate and compare numbers of Tag-specific CD8⁺ T lymphocytes induced against multiple epitopes following Tag immunization, C57BL/6 mice were immunized with syngeneic Tag-transformed B6/WT-19 cells and splenic lymphocytes were analyzed ex vivo at 9, 17, and 90 days postimmunization. Control immunizations were performed using syngeneic B6/T122B1 cells which express a Tag derivative containing alanine substitutions, N210A, N227A, F408A, and N493A, which alter critical MHC class I anchor residues in epitopes I, II/III, IV, and V, respectively. B6/T122B1 cells are not lysed by CTL clones specific for Tag epitopes I, II/III, IV, or V or by bulk SV40 Tag-specific CTL and fail to induce CTL specific for Tag epitopes I, II/III, IV, or V (data not shown) following immunization of C57BL/6 mice.

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Direct enumeration by tetramer staining of SV40 Tag-specific CD8+ cells induced during a primary response to Tag-transformed cell immunization. Pooled splenocytes obtained 9 days following immunization of groups of C57BL/6 mice with transformed cells which expressed either the wild-type Tag (B6/WT-19) or an epitope-negative substituted derivative (B6/T122B1) were stained ex vivo or following secondary in vitro restimulation (Cultured) with anti-CD8 plus Db/I Tet, Kb/IV Tet, or Db/V Tet (A) or Db/II/III (B). Panels A and B represent two different experiments. The specificity of staining by Db/III/III Tet was verified by staining of unimmunized C57BL/6 mice in separate experiments (results not shown). FACS dot plots represent 100,000 events (Ex vivo) or 10,000 events (Cultured).

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Induction and maintenance of Tag-specific CD8+ lymphocytes following immunization with Tag-transformed cells. Groups of three C57BL/6 mice were immunized with transformed cells which expressed either the wild-type Tag (B6/WT-19) or an epitope-negative substituted derivative (B6/T122B1). Spleens were harvested at 9, 17, or 90 days postimmunization, and lymphocytes were pooled and analyzed by FACS analysis following staining with anti-CD8 and Tag-specific tetramers (Db/I Tet, Db/II/III Tet, Kb/IV Tet, and Db/V Tet) or a control tetramer (Kb/gB Tet). Results are presented as the number of CD8+ Tet+ cells per spleen. Epitope II/III-specific T-cell responses were not determined at 17 days. Staining with control tetramers routinely ranged between 0.8 × 104 and 1.8 × 104 CD8+ T cells per spleen in these experiments (D).

Quantitation of SV40 Tag-specific CD8⁺ lymphocytes following immunization with SV40. Footpad immunization with live SV40 results in a nonpermissive infection which leads to Tag synthesis and induction of SV40 Tag-specific CTL (43). The Tag epitope specificities represented in CD8⁺ T lymphocytes of the popliteal lymph nodes or spleen following SV40 immunization via the footpad route have not been characterized. Using the tetramer reagents, lymphocytes obtained from the popliteal lymph nodes or spleens were analyzed ex vivo for the presence of Tag-specific CD8⁺ T lymphocytes 5 or 7 days following footpad immunization with SV40 (Table 1). Kb/IV Tet-staining CD8⁺ T lymphocytes were readily detected in lymphocytes obtained from the popliteal lymph nodes at either 5 or 7 days postimmunization and in splenic lymphocytes obtained 7 days postimmunization (Table 1). Epitope I- or II/III-specific CD8⁺ T lymphocytes were not readily detected above background at either time point by ex vivo staining with the tetramer reagents (Table 1). However, the presence of small numbers of epitope I- and II/III-specific CD8⁺ T cells could not be ruled out due to high levels of background staining in this experiment, as evidenced by staining of 0.5 to 1.1% of CD8⁺ T cells with the control Kb/gB tetramer (Table 1). In fact, IFN- staining of the same populations did reveal the presence of low levels of epitope I- and II/III-specific CD8⁺ T lymphocytes in the spleens but not in the popliteal lymph nodes at 7 days postimmunization (Table 1). Secondary in vitro restimulation and subsequent cytotoxicity assays revealed that epitope I- and II/III-specific CTL were expanded from both popliteal and splenic lymphocyte pools (data not shown). Epitope V-specific CD8⁺ T lymphocytes were not detected by the use of tetramers or cytotoxicity assays. These results reveal that the immunological hierarchy among Tag epitopes is exaggerated in favor of epitope IV in Tag-specific CD8⁺ T-lymphocyte responses induced by SV40 infection compared to immunization with Tag-transformed cells.

View this table: [in this window] [in a new window]   TABLE 1.   MHC class I tetramer staining and peptide-induced intracellular accumulation of IFN- for lymphocytes recovered from SV40-immunized C57BL/6 micea

Quantitation of SV40 Tag-specific CD8⁺ lymphocytes following immunization with rVVs expressing SV40 Tag or Tag epitope minigenes. Immunization with SV40 Tag by systemic infection with rVV-941T, which expresses the full-length Tag, results in induction of CTL specific for immunodominant epitopes I, II/III, and IV (26, 48). Since VV can replicate in the murine host, this system provides the potential for enhanced host cell production of Tag and Tag epitopes compared to SV40 immunization. Groups of C57BL/6 mice were immunized with rVV-941T, and splenic lymphocytes were analyzed ex vivo at 9, 16, and 90 days postimmunization for the presence of Tag-specific CD8⁺ cells by tetramer staining or staining for intracellular IFN- accumulation (Fig. 4A to D). Consistent with results obtained following immunization with Tag-transformed cells or SV40, immunization with rVV-941T induced levels of epitope IV-specific CD8⁺ cells which could be readily detected at 9 days postimmunization by either tetramer staining or IFN- accumulation (Fig. 4D). These epitope IV-specific CD8⁺ cells declined in number but persisted up to 90 days postimmunization at 2.9 × 10⁵ cells per spleen, which represents approximately 40% of the original levels detected on day 9. By comparison, epitope I-specific CD8⁺ cells were detected at low levels at 9 and 16 days postimmunization and represented <10⁴ cells by 90 days (Fig. 4A), which was the limit of detection for these experiments. Epitope II/III-specific CD8⁺ T cells were not detected at 9 days postimmunization, were detected minimally at 16 days postimmunization using Db/II/III Tet, but were not detected ex vivo at 90 days postimmunization (Fig. 4B). Epitope V-specific CD8⁺ cells were not detected in splenocytes from rVV-941T-immunized mice (Fig. 4C). Parallel cytoxicity assays of secondary in vitro-restimulated splenocytes demonstrated that CTL specific for Tag epitopes I, II/III, and IV could be expanded from rVV-941T-immunized animals at each time point.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Analysis of Tag-specific CD8+ lymphocytes ex vivo using tetramers or anti-IFN- antibody at various times after immunization with rVVs which express Tag or Tag epitope minigenes. Groups of three C57BL/6 mice were immunized in the tail vein with 107 PFU of rVVs which express SV40 Tag (rVV-941T) (A to D) or Tag epitope minigenes (I, II/III, V, or IV) appended to an endoplasmic reticulum secretory sequence (E to H). Splenocytes harvested at 9, 16, or 90 days were pooled and analyzed by FACS analysis following staining with Tag epitope-specific MHC class I tetramers (epitopes I, II/III, V, or IV) or for accumulation of intracellular IFN- following a 6-h incubation in the presence of the corresponding synthetic peptides. Results are presented as the number of tetramer-stained CD8+ cells per spleen. The values shown have been corrected for nonspecific staining by subtraction of the number of CD8+ cells detected with control tetramers. Likewise, the number of CD8+ IFN+ cells which stained in response to control peptide was subtracted from the number of CD8+ IFN+ cells which stained in response to specific peptide.

Induction and enhancement of CD8⁺ T lymphocytes specific for the immunorecessive epitope V in Tag. Epitope V in SV40 Tag has been characterized as immunorecessive because immunization with wild-type Tag fails to induce epitope V-specific CTL but immunization with syngeneic cells expressing a Tag which lacks functional epitopes I, II/III, and IV results in induction of epitope V-specific CTL (41, 50). Db/V Tet was used to directly enumerate epitope V-specific CD8⁺ cells 8 days following immunization of C57BL/6 mice with B6/T116A1 cells, which express a Tag derivative lacking epitopes I, II/III, and IV. Db/V Tet stained 0.93% of CD8⁺ T lymphocytes ex vivo (Fig. 5A). Similar levels of epitope V-specific CD8⁺ cells were detected by staining for intracellular accumulation of IFN- following stimulation with the epitope V peptide (data not shown). Epitope V-specific CD8⁺ T lymphocytes expanded to 19.7% of CD8⁺ T cells following secondary in vitro restimulation (Fig. 5A).

View larger version (71K): [in this window] [in a new window]   FIG. 5.   Direct enumeration of Tag epitope V-specific CD8+ cells ex vivo. (A and B) Groups of C57BL/6 mice were immunized with B6/T116A1 cells once (A) (8-day primary) or twice (B) (1 month; 7-day boost); B6/T116A1 cells express a Tag derivative in which epitopes I, II/III, and IV have been deleted or inactivated by alanine substitutions but epitope V has been retained. Pooled splenocytes were analyzed ex vivo or following secondary in vitro restimulation (Cultured) by FACS following staining with tetramers specific for Tag H2-Db-restricted epitopes I or V. (C) Groups of C57BL/6 mice were immunized and boosted with either B6/WT-19 or B6/T116A1 cells after 1 or 3 months as indicated. Splenocytes were harvested after 7 days, pooled, and analyzed by FACS analysis after being stained with the indicated tetramers. Dot plots illustrate results for 200,000 (Ex vivo) or 50,000 (Cultured) cells. Numbers shown in the upper right quadrant correspond to the percentage of CD8+ cells stained by the respective tetramer.

Repertoire of TCR usage by SV40 Tag-specific CD8⁺ cells in vivo. The diversity of TCR usage by CD8⁺ T cells responding to SV40 Tag epitopes has been largely uncharacterized. In initial attempts to investigate the TCR repertoire used by Tag-specific CD8⁺ T lymphocytes, Tag-specific CTL clones were analyzed for TCR subunit usage by FACS analysis (Table 2). The results revealed that CTL clones specific for epitopes I and II/III expressed TCR10 whereas selected epitope I- and V-specific CTL clones expressed TCR7. Sequence analysis confirmed that distinct CDR3 sequences were expressed by CTL clones of differing epitope specificities which used similar TCR segments (data not shown). Curiously, all three epitope II/III-specific CTL clones utilized TCR10; although CTL clones K-19 and Y-2/Y-3 were isolated in independent experiments, they express identical CDR3 sequences (data not shown). This suggested the presence of restricted TCR usage in the development of epitope II/III-specific CTL responses. Epitope V-specific CTL clones utilized TCR7, TCR9, or TCR2, while epitope IV-specific CTL clones utilized TCR9, TCR8, or TCR5. These results reveal similarity and diversity in TCR usage by SV40 Tag-specific CTL clones which were established following immunization with Tag-transformed cells.

View this table: [in this window] [in a new window]   TABLE 2.   Specificity, MHC restriction, and TCR usage for SV40 Tag-specific CTL clones

View larger version (28K): [in this window] [in a new window]   FIG. 6.   TCR usage by Tag-specific CD8+ cells. CD8+ cells were analyzed for Tag specificity by tetramer staining and for TCR expression by FACS analysis. The results are presented as the percentage of CD8+ Tet+ lymphocytes stained with a TCR-specific antibody. (A) Staining of splenocytes ex vivo from C57BL/6 mice 9 days after immunization with B6/WT-19 cells using anti-CD8 antibody, Kb/IV Tet, and TCR-specific monoclonal antibodies. (B to D) Splenocytes were harvested from C57BL/6 mice immunized for 1 month with B6/WT-19 cells and restimulated in vitro with irradiated B6/WT-19 cells for 5 days. Cells were triple stained using anti-CD8 antibody, tetramer (Kb/IV Tet [B], Db/I Tet [C], and Db/II/III Tet [D]), and a panel of TCR-specific antibodies and analyzed by flow cytometry. (E) Splenocytes were harvested from C57BL/6 mice immunized with B6/T116A1 cells (1 month followed by a 7-day boost) and restimulated in vitro with irradiated B6/WT-19 cells for 5 days. Cells were triple stained using anti-CD8 antibody, Db/V Tet, and TCR-specific antibodies and analyzed by flow cytometry. The most prominent TCR chains used are indicated by the corresponding TCR number on top of each bar. Asterisks indicate TCR segments used by the SV40 Tag-specific CTL clone(s) with corresponding epitope specificity.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The direct analysis of CD8⁺ T lymphocytes from Tag-immunized C57BL/6 mice clearly demonstrates that immunodomination by epitope IV is established in vivo. In addition, the role of epitope V as a true immunorecessive epitope was reinforced by the finding that epitope V-specific CD8⁺ T cells were undetectable by ex vivo analysis of lymphocytes from C57BL/6 mice immunized with wild-type Tag, even following a priming-and-boosting protocol. This indicates that epitope V-specific T-lymphocyte responses are controlled in vivo rather than at the level of in vitro expansion. In contrast, in the absence of immunodomination by the other Tag epitopes, epitope V-specific CD8⁺ T lymphocytes were recruited in vivo following immunization with a cell line expressing only epitope V or an rVV-expressing epitope V as a minigene. Boosting of the epitope V-specific response with epitope V-only cells resulted in further expansion of epitope V-specific CD8⁺ T cells in vivo, indicating that epitope V-specific CD8⁺ T cells can be recruited to high levels by using an appropriate immunization strategy.

Frequency measurements of Tag epitope-specific CD8⁺ T lymphocytes in Tag-immunized C57BL/6 mice obtained by staining with epitope-specific MHC class I tetramers or by intracellular IFN- accumulation revealed significantly larger numbers of epitope-specific CD8⁺ T cells than were estimated by limiting-dilution analysis. For example, previous estimates of CTL precursor frequencies indicated that 1 in 14,000 splenocytes from 9-day B6/WT-19-immunized C57BL/6 mice were specific for Tag epitope IV (41). Considering that CD8⁺ T lymphocytes comprised roughly 10% of splenic lymphocytes, epitope IV-specific CTL would represent approximately 1 in 1400 CD8⁺ T cells. In contrast, the direct methods used in the present study indicated that 11% (or ~1 in 10) of CD8⁺ cells were specific for Tag epitope IV. Frequencies determined for CD8⁺ T cells specific for Tag epitopes I (1 in 32) and II/III (1 in 82) also were approximately 100-fold higher than those previously estimated by limiting-dilution analysis. This overall increase in sensitivity of approximately 100-fold is in line with similar studies in other systems (14, 25, 39, 44). Even with improved sensitivity, however, epitope V-specific CD8⁺ T lymphocytes were not detected ex vivo following immunization with full-length Tag. It is unclear how Tag-specific CTL responses might differ if Tag were produced by a chronic viral infection. In other commonly studied models of immunodominance, e.g., lymphocytic choriomeningitis virus, frequencies of epitope-specific CD8⁺ T cells can approach 1 in 3 shortly after infection (39). This difference might be attributed to the extensive proliferation of lymphocytic choriomeningitis virus in the spleen and lymph nodes (9) compared to the proliferative responses generated following Tag immunization in this study. We note that the frequency of memory T cells detected for the immunodominant epitope IV by 90 days postimmunization was similar following immunization with either B6/WT-19 cells, rVV-941T, and rVV-ESIV and is in line with the number of memory T cells detected in other systems (13, 39).

Three distinct vehicles were used to immunize mice with full-length SV40 Tag, including Tag-transformed cells, SV40, and rVV-941T. Specific mechanisms which deliver Tag epitopes into the MHC class I antigen-processing pathway will probably vary for each of these vehicles due to their ability to target different cell types and due to the use of distinct routes of immunization. Induction of Tag-specific CTL by immunization with Tag-transformed cells is likely to rely on re-presentation, or cross-priming, of the antigen by professional antigen-presenting cells (APCs) (29, 55). Cross-priming also may be utilized in cases where rVV-941T or SV40 infect cells that do not express costimulatory molecules. Tag delivery through immunization with SV40 or rVV-941T, however, may result in infection of APCs which directly sensitize CD8⁺ T cells. For example, SV40 transforms macrophages (24, 37). SV40 establishes a nonpermissive infection in mouse cells, with the virus cycle limited to the expression of Tag, thus inducing the proliferation of infected cells which persist until they are eliminated by immune mechanisms (1, 35). In contrast, VV replicates and releases new virus progeny, which in turn can infect more cells (38), resulting in increased availability of Tag.

On the other hand, immunization with Tag-transformed cells has the potential for rapid delivery of a relatively large amount of presynthesized Tag protein and/or preprocessed Tag epitopes for cross-priming. In fact, immunization with either SV40 or rVV-941T resulted in exaggeration of the immunodominance of epitope IV over epitopes I and II/III compared to that found for immunization with B6/WT-19 Tag-transformed cells. The requirement for host cell biosynthesis of Tag might limit the availability of H2-D^b-restricted Tag epitopes in vivo and therefore might skew the immunodominance of Tag-specific CD8⁺ T-cell responses in favor of epitope IV. Although the precise mechanisms by which Tag epitopes are derived from each vehicle are likely to differ, it is important to note that the hierarchy observed for induction of Tag epitope-specific CD8⁺ cells in each case was consistent. Thus, epitope IV-specific CD8⁺ T lymphocytes were clearly dominant regardless of the mode of immunization, while epitope V-specific CD8⁺ T lymphocytes were never detected following immunization with wild-type Tag.

Deficiencies in antigen processing of selected T-cell epitopes contribute to immunogenicity and rank within the immunological hierarchy of T-cell responses (18, 58). Specific mechanisms which might lead to the immunorecessive phenotype include inefficient processing of the epitope from the native protein, inefficient transport by transporters associated with antigen processing (TAP), or relatively weak interactions with MHC class I molecules compared to immunodominant epitopes. The amino acid context surrounding epitope V within Tag limits processing, presentation, and immunogenicity for at least one test epitope (40). By extension, the results of that study imply that the immunogenicity of epitope V may be limited by similar constraints on processing of the epitope. In addition, epitope V is destroyed by the proteasome when expressed as a minigene from an rVV (26). This phenotype can be rescued by the use of inhibitors of the proteasome, addition of alanine residues at either terminus of the minigene, or targeting the peptide to the endoplasmic reticulum using ES, suggesting that the epitope V peptide is readily degraded in the cytosol. We have previously shown that the epitope V peptide functions efficiently in TAP assays, but it forms relatively unstable complexes with H2-D^b molecules compared to epitopes I and II/III (26). Thus, relatively small numbers of D^b/V complexes on the APC surface might limit the induction of epitope V-specific CD8⁺ T lymphocytes in the presence of immunodominant Tag epitope-specific CD8⁺ T lymphocyte responses. The number of epitope D^b/V complexes on Tag-expressing cells remains to be determined.

The abundance of a particular peptide-MHC complex, however, is not necessarily the determining factor in establishing the hierarchy of CTL responses. In fact, Pamer and coworkers (13, 56) have shown that the hierarchy of the CD8⁺ T-cell responses to three H2-K^d-restricted epitopes from Listeria monocytogenes is inversely related to the abundance of the epitopes. The ability of epitope V to induce the accumulation of high levels of cognate CD8⁺ T lymphocytes in vivo following immunization and boosting with the epitope I-, II/III-, and IV-deficient Tag derivative suggests that the immunorecessive status of epitope V is not due solely to the biophysical properties of the peptide or Tag context alone. In fact, at 17 to 25% of CD8⁺ cells, frequencies for epitope V-specific CD8⁺ T cells were obtained in vivo which approximated the optimal frequencies observed for epitope IV- or I-specific CD8⁺ T cells obtained following two immunizations with B6/WT-19 cells. Thus, the number of CD8⁺ T-cell precursors specific for epitope V can readily expand under the proper circumstances, suggesting that limitations in the response to epitope V are not due to an inherent inability of epitope V-specific T-cell precursors to expand in vivo. Although the basis for the immunorecessive nature of the epitope V-specific CD8⁺ T-lymphocyte response remains to be determined, our results support an important role for immunodomination by other Tag epitope-specific CD8⁺ T cells.

Analysis of TCR usage has been used to estimate the variability of epitope-specific T-lymphocyte responses. Studies investigating the variability of the TCR usage by T lymphocytes responding to immunodominant epitopes have shown that both limited (2, 17, 19, 21, 22) and highly variable (20, 30, 57) usage can be detected, depending on the system studied. A few studies have indicated that the TCR usage by CTL specific for immunodominant epitopes is more diverse than the TCR usage by CTL specific for subdominant epitopes within the same system (10, 16). Thus, while general conclusions regarding the role of TCR diversity in immunodominance cannot be made, it remains to be determined whether the variability of the response might contribute to immunodominance in some systems. The advent of MHC class I tetramers and intracellular cytokine staining has made it possible to analyze the TCR repertoire of epitope-specific CD8⁺ T cells ex vivo or after a brief period of in vitro expansion instead of needing multiple rounds of in vitro restimulation or relying on the development of CTL clones. Using these approaches, analysis of TCR usage by epitope-specific CD8⁺ T cells indicates that the response to immunodominant epitopes is characterized by overall diversity but with preferential expansion of T cells expressing a few TCR subunits (12, 18, 49).

Direct counting of Tag epitope-specific CD8⁺ T lymphocytes by tetrameric reagents has made it possible to examine the TCR repertoires responding to the individual Tag epitopes. The results of this analysis indicate that the TCR repertoire varied in both the identity of the predominant TCR subunit(s) as well as the overall diversity of TCR chains utilized within each response. The results obtained by the analysis of Tag-specific CD8⁺ populations from mice immunized with Tag-transformed cells or following a single period of secondary in vitro restimulation appear to correlate well with the results obtained by the analysis of established Tag-specific CTL clones. Notable exceptions appear to be the use of TCR13 and TCR8 in CD8⁺ populations responsive to epitopes IV and I, respectively. Analysis of additional CTL clones may reveal their usage, unless expansion of CD8⁺ lymphocytes expressing these TCR is somehow limited in vitro.

Favored usage of TCR5 and TCR8 by epitope IV-specific CD8⁺ T cells was not unique to the particular methods used for induction or expansion, which included priming, boosting, and in vitro restimulation with Tag-transformed syngeneic cells. We did not observe narrowing of the CD8⁺ response to a particular TCR subunit following boosting of the Tag response as has been reported for CD8⁺ responses to a Listeria monocytogenes epitope (11, 12). Additionally, the fact that epitope IV-specific CTL clones and freshly isolated epitope IV-specific CD8⁺ T cells utilize similar TCR chains indicates that in vitro cultivation did not bias the outgrowth of clones using less frequently represented TCR subunits from the in vivo CD8⁺ population as suggested for other systems (49). TCR usage by CD8⁺ T lymphocytes specific for Tag epitopes I and V was similarly diverse, suggesting that the immunorecessive nature of the epitope V-specific CD8⁺ T-lymphocyte response is not due to constraints on TCR diversity.

In contrast to the diverse TCR repertoire utilized in response to epitope I, IV, and V, epitope II/III-specific CD8⁺ T lymphocytes and CTL clones appear to utilize TCR10 exclusively. Results of CDR3 nucleotide sequence analysis revealed identity among TCR subunits used by epitope II/III-specific CTL clones Y-2, Y-3, and K-19 (data not shown). Together, these results imply that the TCR repertoire of Tag epitope II/III-specific CD8⁺ T lymphocytes may be very limited. The variability of the TCR repertoire in this CD8⁺ T-cell population remains to be determined. Mechanisms which have been suggested to limit the diversity of a specific T-cell response include the deletion or inactivation of epitope-specific T cells due to cross-reactivity with self antigens (19), limitations in the efficiency with which epitopes are presented for recognition by naive CD8⁺ T cells (16), and a delay in the relative time of initial antigen exposure which might limit the expansion of antigen-specific T cells (8). It remains to be determined whether the diversity of TCR usage by epitope II/III-specific CD8⁺ T cells would be altered in the absence of ongoing CD8⁺ T-cell responses to epitopes I and IV or whether the limited TCR diversity observed for epitope II/III-specific CD8⁺ T cells may explain the less vigorous response to this epitope compared to the responses to epitopes I and IV.

The results of this study clearly demonstrate that the hierarchy of Tag epitope-specific CD8⁺ T-cell responses is established in vivo. Although the specific mechanisms which lead to immunodomination in this system remain to be determined, the advent of methods for direct detection of responding T cells has provided powerful tools for the investigation of the hierarchical relationships established in vivo, and their use should further enhance our understanding of immunodomination.