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Sequential Loss of Cytotoxic T Lymphocyte Responses to Simian Virus 40 Large T Antigen Epitopes in T Antigen Transgenic Mice Developing Osteosarcomas¹

Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [T. D. S., S. S. T.], and The Jackson Laboratory, Bar Harbor, Maine 04609 [B. B. K.]

	ABSTRACT

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The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2^b). 501 mice express SV40 T ag under the influence of the -amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2^b-restricted T ag CTL epitopes, which include epitope I (T ag 206–215), epitope II/III (T ag 223–231), the immunorecessive epitope V (T ag 489–497), restricted by H2-D^b, and epitope IV (T ag 404–411), restricted by H2-K^b. We demonstrate that 501 mice are partially tolerant to the H2^b-restricted T ag epitopes. Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-K^b-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4⁺ T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV were detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8⁺ cells derived from 3-month-old 501 mice with H2-K^b/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8⁺ cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-K^b/epitope IV tetramer staining CD8⁺ cells. Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.

	INTRODUCTION

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Establishment and progression of tumors in the immunocompetent host induced by the dominant oncogene, T ag³ encoded from the SV40 early region, is influenced by the immune response to MHC class I-restricted T-cell epitopes (1) . In high-responder mouse strains, T ag-transformed cells become transplantable but only as a result of down-regulation of MHC class I molecules and upon prolonged culture in vitro. In T ag transgenic mice, however, tumors grow progressively and at times metastasize to distant sites (2, 3, 4, 5, 6, 7) . Depending upon the tissue expression of the transgene, immunosurveillance by CD8⁺ T cells is compromised as a result of the development of immunological tolerance to T ag CTL epitopes (8, 9, 10, 11) . In instances where T ag expression is driven by the SV40 early promoter, transgene expression in the thymus leads to the induction of central tolerance because of clonal deletion of CD8⁺ T cells specific for T ag CTL epitopes, allowing the progressive growth of choroid plexus tumors (8 , 10 , 12) . However, in a number of model systems, the expression of T ag or T ag fragments in the periphery has had diverse effects on the host T-cell repertoire, ranging from no apparent loss of reactivity (13, 14, 15) to the induction of various levels of nonresponsiveness (11 , 16 , 17) .

The cellular immune response in the C57BL/6 high-responder strain of mice is characterized by the generation of CTLs against three immunodominant epitopes, designated epitope I (amino acid residues 206–215), epitope II/III (amino acid residues 223–231), and epitope IV (amino acid residues 404–411; Refs. 18, 19, 20, 21 ). Epitopes I and II/III are H2-D^b restricted, whereas epitope IV is H2-K^b restricted. In addition, CTLs against the immunorecessive H2-D^b-restricted epitope V (amino acid residues 489–497) are generated after immunization with T ag variants that lack the three immunodominant T ag epitopes (22) . We have demonstrated recently that induction of CTLs specific for the H2-K^b-restricted epitope IV leads to control of spontaneous T ag-induced choroid plexus tumors in T ag transgenic mice upon X-irradiation and reconstitution with spleen cells from normal C57BL/6 mice (10) , indicating a role for this H2-K^b-restricted T ag epitope in the control of endogenous tumor progression.

Mice of the 501 (H2^b) lineage express SV40 T ag as a transgene from the -amylase promoter, which leads to T ag expression in salivary glands as well as bone (5) . T ag expression in osteoblasts results in the development of osteogenic osteosarcomas in bones of the axial skeleton as well as the femur and humerus. Metastases to the liver are detected frequently and are also found occasionally in the lungs. Expression of T ag in the salivary glands is detected by 3 months of age with increased expression by 6 months of age, although no neoplasia develops in the salivary glands.⁴ Osteosarcomas are first detected by 8 months of age in 501 mice, which have an average life span of 13 months of age. The CTL response to individual H2^b T ag epitopes as well as the kinetics of tolerance onset to these CTL epitopes in 501 mice has not been investigated previously.

In this report, we determined whether 501 mice develop CTL precursors that can be activated against the four defined H2^b T ag epitopes both early and late after the onset of T ag expression in the periphery. The results indicate that although 4-month-old 501 mice maintained CTL precursors capable of responding to epitopes I, IV, and V, older mice developed increased tolerance to the T ag CTL epitopes. Loss of CTL responsiveness, however, varied for each epitope. In particular, loss of responsiveness to epitope IV correlated directly with the appearance of T ag-expressing osteosarcomas.

	MATERIALS AND METHODS

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Mice.
Male C57BL/6 (H2^b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in isolator cubicles at the animal facility of the Milton S. Hershey Medical Center. Line C57BL/6-TgN(Amy1T ag)501Knw, or 501, mice express full-length SV40 T ag as a transgene under the influence of the -amylase promoter and have been described previously (5) . 501, 501/CD4^-/- knockout, and B6/CD4^-/- (23) mice were bred and maintained at The Jackson Laboratory and at the animal research facility of the Milton S. Hershey Medical Center.

Cell Lines and Media.
B6/WT-19 is an SV40 transformed C57BL/6 mouse embryo fibroblast line that expresses wild-type T ag (24) . B6/K-1,4,5 was derived from a T ag-transformed C57BL/6 kidney cell line by sequential immunoselection with T ag-specific CTL clones and lacks the T ag epitopes I, II/III, IV, and V (18 , 21 , 25 , 26) . 501/2484 col 3 was derived from an osteosarcoma of a 501 mouse and expresses full-length T ag (5) . All T ag-transformed cell lines were maintained in DMEM supplemented with 100 units of penicillin/ml, 100 µg of streptomycin/ml, 100 µg of kanamycin/ml, 2 mM L-glutamine, 10 mM HEPES buffer, 0.075% (w/v) NaHCO₃, and 5–10% FBS. RMA (H2^b) cells (27) were maintained in suspension using RPMI 1640 supplemented with 10% FBS, 100 units of penicillin/ml, 100 µg of streptomycin/ml, 2 mM L-glutamine, and 50 µM 2-mercaptoethanol.

Viruses and Synthetic Peptides.
rVVs used in this study have been described previously (28 , 29) and include rVV-941T, which encodes full-length SV40 T ag, a series of rVVs encoding T ag epitope minigenes designated rVV-I (T ag sequence 206–215), rVV-II/III (T ag 223–231), rVV-IV (T ag sequence 404–411), and rVV-VAA (T ag 489–497 plus 2 Ala at the COOH terminus) and a corresponding series of minigenes with a preceding ES and designated as rVV-ES I, rVV-ES II/III, rVV-ES IV, and rVV-ES V. The wild-type vaccinia virus strain WR (VV-WR; ATCC VR-119), from which all of the rVVs were derived, was used in these experiments. All peptides used were synthesized at the Macromolecular Core Facility of the Milton S. Hershey Medical Center by FMoc chemistry using an automated peptide synthesizer (9050 MilliGen PepSynthesizer). Peptides were solubilized in DMSO and diluted to the appropriate concentration with RPMI 1640. Peptides used in these experiments correspond to SV40 T ag epitopes I (SAINNYAQKL), II/III (CKGVNKEYL), IV (VVYDFLKC), and V (QGINNLDNL) as well as the optimized H2-D^b binding peptide D^bN5 (SMIKNLEYM; Refs. 30 and 31 ) and the H2-K^b-restricted peptide gB498–505 (SSIEFARL; Ref. 32 ) from HSV.

Immunization of Mice and in Vitro Restimulation of Bulk CTLs.
Mice were immunized at the indicated ages by i.p. injection of 2–5 x 10⁷ T ag-transformed cells or by i.v. injection of 1 x 10⁷ pfu rVVs. Mice were sacrificed at 2–4 weeks after immunization, and single-cell suspensions of RBC-depleted spleen cells were restimulated in vitro with gamma-irradiated B6/WT-19 cells as described (30) . Briefly, 1 x 10⁷ spleen cells from immunized animals were mixed with 5 x 10⁵ gamma-irradiated (10,000 rads) B6/WT-19 cells in 4 ml of complete RPMI 1640 supplemented with 10% FBS/well of a 12-well tissue culture plate. To verify vaccinia virus infection, 1 x 10⁷ spleen cells from each vaccinia virus-infected mouse were restimulated in vitro with 5 x 10⁵ irradiated VV-WR-infected C57BL/6 spleen cells in 4 ml of complete RPMI 1640 supplemented with 10% FBS/well of a 12-well plate. VV-WR-infected C57BL/6 spleen cell stimulators were prepared by infection of naive C57BL/6 spleen cells (1 x 10⁷ per ml PBS/BSA) with VV-WR (multiplicity of infection, 10) for 1 h at 37°C with occasional agitation, followed by a 3-h incubation in complete RPMI 1640 at 37°C, 5% CO₂. Infected cells were gamma-irradiated (60,000 rads) and washed free of virus before use.

Maintenance of CTL Clones.
SV40 T ag-specific CTL clones used in this study were maintained by in vitro passage as described previously (20) and include clones K-11, K-19 (33 , 34) , Y-4 (18) , and H-1 (30) , which recognize T ag epitopes I (residues 206–215), II/III (residues 223–231), IV (residues 404–411), and V (residues 489–497), respectively.

Cytotoxicity Assays.
Assays for CTL lysis were performed on day 6 after in vitro restimulation as described previously (10) . Statistical analysis of responders versus nonresponders in development of CTLs in tumor-bearing and tumor-free 501 mice was performed using Fisher’s exact test with a two-sided P < 0.01 considered significant using Instat (GraphPad Software).

Histology and Immunohistochemistry.
For histology and immunohistochemistry, mice were perfused with 10% formalin, and tissues were fixed with 10% formalin for an additional 24 h. Tissues were decalcified for 8 h prior to embedding in paraffin blocks. Seven-µm blocks were cut on a microtome and collected onto positively charged slides. Parallel sections were stained by H&E or immunohistochemistry for T ag as described previously (10) .

X-Rays.
X-rays of mice were performed by the trained veterinary staff of the Department of Comparative Medicine at the Milton S. Hershey Medical Center. Mice were anesthetized immediately prior to X-ray and were placed in a dorsoventral position on a Kodak MIN-R2 cassette (Eastman Kodak, Rochester, NY) containing Kodak MIN-R film. X-rays were performed using 400 mAmps and 58 kV for 0.015 s.

Preparation of Class I MHC Tetramers, Staining of Epitope-specific T Cells, and Flow Cytometry.
Production and characterization of the H2-K^b/T ag epitope IV (K^b/IV Tet) and H2-K^b/HSV gB498–505 (K^b/gB Tet) tetramers were achieved essentially as described (35) and will be described in detail elsewhere.⁵ PE-labeled K^b/IV Tet used in this study was prepared using an analogue of the native epitope IV peptide containing a C411L substitution (VVYDFLKL), which enhances the stability of this peptide with H2-K^b without disrupting T-cell recognition (data not shown). For staining of lymphocyte populations, RBC-depleted splenocytes were incubated with rat antimouse CD16/CD32 (PharMingen) and 50 µg/ml streptavidin (Molecular Probes) for 30 min on ice to block Fc receptors and nonspecific binding of streptavidin conjugated tetramers, respectively. After a single wash, spleen cells were incubated with phycoerythrin-labeled tetramers and FITC-labeled rat antimouse CD8a (53-6.7; PharMingen) for 1 h on ice. Cells were fixed with 2% paraformaldehyde and analyzed using a FACScan (Becton Dickinson, San Jose, CA) flow cytometer, and data were analyzed and prepared using CELLQuest software (Becton Dickinson). The percentage of CD8⁺ cells that stained specifically with K^b/IV Tet was determined by subtracting the percentage of CD8⁺, K^b/gB Tet⁺ cells from the percentage of CD8⁺, K^b/IV Tet⁺ cells within the same population.

Intracellular Cytokine Assay.
For staining of intracellular IFN-, spleen cells were harvested from rVV-ES IV immunized mice at 14 days after immunization. RBC-depleted spleen cell suspensions were prepared as above, and 5 x 10⁶ spleen cells were incubated with 1 µM of the indicated synthetic peptides representing T ag or control epitopes and 1 µg per ml brefeldin A in 2 ml of complete RPMI 1640 containing 10% FBS/well of a 24-well plate for 6 h at 37°C, 5% CO₂. Cells were stained for intracellular IFN- using the Cytofix/Cytoperm kit (PharMingen) according to the manufacturer’s specifications. Briefly, stimulated cells were washed twice, and then Fc receptors were blocked by incubation with rat antimouse CD16/CD32 (PharMingen) for 20 min, followed by staining with phycoerythrin-labeled rat antimouse CD8 (PharMingen) for 30 min. After fixation and permeabilization for 20 min, cells were stained with FITC-labeled rat antimouse IFN- (PharMingen) or an isotype control antibody for 30 min and then analyzed by flow cytometry as described above. The percentage of CD8⁺ cells that express intracellular IFN- was calculated by subtracting the percentage of cells that stained nonspecifically with the isotype control antibody from those that stained specifically for IFN- after stimulation with the specific T ag epitope, and by further subtracting those cells that stained positive for IFN- after incubation with an unrelated peptide.

	RESULTS

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Tumors from 501 Transgenic Mice Present Multiple T ag Epitopes for CTL Recognition.
Mice of the 501 lineage develop osteogenic osteosarcomas. These tumors consist of fibrous sheets of neoplastic mesenchymal cells (Fig. 1 A, panel a; Fig. 1 B, panel c) and are associated with osteoid and fibrous connective tissue production (Fig. 1 A, panel c; Fig. 1 B, panel a). Staining for T ag expression within tumors from distinct anatomical sites and from multiple animals revealed the presence of T ag-expressing cells throughout these tumors (Fig. 1, A and B , panels b and d). Because expression of SV40 T ag in the periphery can lead to the induction of tolerance or anergy to T ag CTL epitopes, we determined whether the four defined H2^b T ag epitopes were expressed by 501 tumors. The osteoblast line 501-2484 col 3, derived from a 501 osteosarcoma, was tested for susceptibility to lysis by a panel of CTL clones specific for each of the four T ag epitopes in a ⁵¹Cr-release assay. 501-2484 col 3 cells were lysed as efficiently as the T ag transformed cell line B6/WT-19 by each of the CTL clones (Fig. 2 ) indicating that epitopes I, II/III, IV, and V are processed and presented by class I MHC molecules of this 501-derived tumor cell line. In addition, C57BL/6 mice immunized with 501-2484 col 3 cells developed CTLs specific for the immunodominant epitopes I, II/III, and IV after in vitro stimulation of spleen cells (data not shown). These results indicate that each of the four previously defined H2^b-restricted T ag epitopes are presented by osteosarcomas from 501 mice.

View larger version (77K): [in this window] [in a new window]   Fig. 1. Osteosarcomas from 501 mice. A, paraffin-embedded sections of an osteogenic osteosarcoma of the rib from a naive 501 mouse, 501-123, stained by H&E (panels a and c) and immunohistochemistry for T ag (panels b and d) showing sheets of mesenchymal cells (panels a and b) and osteoid production (panels c and d). B, staining of paraffin-embedded sections from osteogenic osteosarcomas of the humerus from mouse 501-34 (panels a and b) and the clavicle from mouse 501-59 (panels c and d) by H&E (panels a and c) and immunohistochemistry for T ag (panels b and d). Both 501-34 and 501-59 failed to develop epitope IV-specific CTLs after immunization with rVV-ES IV (see Figs. 6 8 , respectively). A, panels a and b: x400, panels c and d, x200; B, x200 (all panels). Staining of nuclear T ag appears brown.

View larger version (40K): [in this window] [in a new window]   Fig. 2. 501 tumor cell lines present four H2b epitopes for CTL recognition. CTL clones specific for T ag epitopes were tested for lysis of 51Cr-labeled B6/WT-19 (wild-type T ag), 501-2484 col 3 and B6/K-1, 4, 5 (epitopes I, II/III, IV, and V loss T ag) cells in a 5-h assay. CTL clones were used at an E:T of 2:1, 5:1, 5:1, and 10:1, respectively, for clones K-11, K-19, Y-4, and H-1.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Immunization of 501 mice with full-length T ag induces epitope IV-specific CTLs. Groups of two to three 4-month-old C57BL/6 (A and C) and 501 (B and D) mice were immunized with 2 x 107 B6/WT-19 cell i.p. (A and B) or 1 x 107 PFU rVV-941T i.v. (C and D). After 2 and 4 weeks, respectively, spleen cells from immunized mice were restimulated in vitro with B6/WT-19 cells for 6 days. Responder cells were tested for their ability to lyse 51Cr-labeled RMA cells pulsed with 1 µM of the peptides representing T ag epitope I (), II/III (•), IV (), V (), or the control epitope DbN5 (), and the T ag-transformed cell lines B6/WT-19 () and B6/K-1, 4, 5 ().

Groups of C57BL/6 and 501 mice of 4 months of age were immunized with 1 x 10⁷ pfu of rVVs encoding each of the T ag epitopes as a minigene, with or without a preceding ES. In the case of epitope V, a minigene construct encoding epitope V followed by two alanine residues was used instead of the epitope V sequence alone, because in the absence of these additional residues, epitope V fails to be immunogenic in C57BL/6 mice when expressed as a minigene (28) . After 4 weeks, spleen cells were restimulated in vitro with B6/WT-19 cells and then tested for their ability to lyse peptide pulsed RMA cells in a ⁵¹Cr-release assay. Immunization of C57BL/6 mice with each rVV resulted in the induction of CTLs specific for the appropriate T ag epitope (Fig. 4, I–P ). In contrast, only immunization with rVV-ES I, rVV-IV, rVV-ES IV, and rVV-ES V induced detectable CTL responses in 501 mice (Fig. 4, A–H ). Thus, 501 mice retain the ability to respond to T ag epitope I, but this response is only detected if the mice are immunized with rVV-ES I (Fig. 4B ). This suggests that a limited number of epitope I-specific CTL precursors are available to respond or that they are anergic and only respond to a strong immunization. Immunization of 501 mice with rVV-II/III or rVV-ES II/III failed to induce epitope II/III-specific CTLs in multiple experiments (Fig. 4, C and D ), suggesting that CTLs specific for this epitope were absent from 501 mice or were unable to respond, even to this strong stimulus. Immunization of 501 mice with rVVs expressing epitope IV resulted in strong epitope IV-specific CTL responses (Fig. 4, E and F ), demonstrating that the relatively decreased responses to epitope IV observed using immunization with full-length T ag (Fig. 2, B and D ) could be enhanced using this approach. Immunization with the rVV-V-AA construct failed to induce a significant response in 501 (Fig. 4G ) compared with C57BL/6 (Fig. 4O ) mice, indicating that the ability of 501 mice to respond to epitope V is also reduced compared with C57BL/6 mice. Immunization of 501 mice with rVV-ES V, however, resulted in the induction of efficient epitope V-specific CTLs (Fig. 4H ). All mice immunized with rVVs developed vaccinia virus-specific CTLs after in vitro stimulation of spleen cells with VV-WR-infected C57BL/6 spleen cells, indicating that vaccinia virus infection of mice had occurred (data not shown). Thus, the limited response of 501 mice to T ag CTL epitopes I, IV, and V can be substantially enhanced by immunization with rVVs expressing T ag epitopes preceded by ES.

View larger version (36K): [in this window] [in a new window]   Fig. 4. rVV-ES T ag epitope minigene immunization induces epitopes I-, IV-, and V-specific CTLs in 501 mice. Groups of two to three 4-month-old 501 (A–H) or C57BL/6 (I–P) mice were immunized i.v. with 1 x 107 pfu of the indicated rVV with or without a preceding ES. After 4 weeks, spleen cells from immunized mice were restimulated in vitro with B6/WT-19 cells for 6 days. Responder cells were tested for their ability to lyse 51Cr-labeled RMA cells pulsed with 1 µM of the indicated peptides.

View larger version (31K): [in this window] [in a new window]   Fig. 5. T ag epitope CTL response in 6-month-old 501 mice. Groups of 501 (A–C) or C57BL/6 (D–F) mice were immunized i.v. with 1 x 107 PFU rVV-ES I (A and D), rVV-ES IV (B and E) or rVV-ES V (C and F). After 4 weeks, spleen cells from individual animals were restimulated in vitro with B6/WT-19 cells for 6 days. Responder cells were tested for their ability to lyse 51Cr-labeled RMA cells pulsed with 1 µM of the indicated peptides representing T ag epitopes, the H2-Db binding peptide DbN5, or the H2-Kb-binding peptide HSV gB 498–505 (gB). The number of mice in each group is indicated.

View larger version (33K): [in this window] [in a new window]   Fig. 6. T ag epitope IV-specific CTL response in 501 mice of increasing age. Groups of two 501 mice were immunized i.v. with 1 x 107 pfu of rVV-ES IV at the indicated ages A–E. After 4 weeks, spleen cells from individual animals were restimulated in vitro with B6/WT-19 cells for 6 days. Responder cells were tested for their ability to lyse 51Cr-labeled RMA cells pulsed with 1 µM of the indicated peptides. Alternatively, spleen cells were restimulated in vitro with VV-WR-infected, gamma-irradiated C57BL/6 spleen cells and tested for the ability to lyse 51Cr-labeled B6/K-1, 4, 5 cells infected with VV-WR or mock infected (C, inset). Arrows, 501 progeny identification numbers and the matching responses. *, 501-34 had an osteosarcoma of the right humerus.

View larger version (84K): [in this window] [in a new window]   Fig. 7. X-ray analysis of 12-month-old 501 mice for the presence of osteosarcomas. Mice were X-rayed in the dorsoventral position as described in "Materials and Methods." A, 501-61 was negative for tumors. B, 501-62 had an osteosarcoma of the left humerus (arrow). C, 501-66 was negative for tumors. D, 501-59 had osteosarcomas on the right ribs and the left scapula and clavicle (arrows).

View larger version (43K): [in this window] [in a new window]   Fig. 8. Tolerance to epitope IV, but not to epitope V, correlates with the appearance of tumors. 501 and C57BL/6 mice of the indicated ages were immunized with 1 x 107 pfu rVV-ES IV (A–E) or rVV-ES V (F–K). Tumor status was determined by X-ray analysis and confirmed by necropsy. After 4 weeks, spleen cells from individual animals were restimulated in vitro with B6/WT-19 cells for 6 days. Responder cells were tested for their ability to lyse 51Cr-labeled RMA cells pulsed with 1 µM of the indicated peptides. Alternatively, spleen cells were restimulated in vitro with VV-WR-infected, gamma-irradiated C57BL/6 spleen cells and tested for the ability to lyse 51Cr-labeled B6/K-1, 4, 5 cells, which were infected with VV-WR or mock infected (B, D, I, and J, insets). The CTL responses shown in A–D correlate with the mice shown in Fig. 7, A–D .

Decreased Numbers of Epitope IV-specific CTLs Are Detected in 501 Mice.
Our results suggested that decreased responsiveness to T ag epitope IV correlates with the appearance of osteosarcomas in 501 mice. This loss of T ag epitope IV-specific CTL reactivity might be explained by loss of epitope IV-specific CTL precursors through deletion or by the induction of anergy among epitope IV-specific CD8⁺ T cells. To determine whether a quantitative difference in epitope IV-specific CD8⁺ T cells existed between tumor-bearing versus tumor-free 501 mice, we used class I MHC tetramers (35) composed of H2-K^b and T ag epitope IV peptide (K^b/IV Tet) to determine the number of epitope IV-specific T cells that developed in rVV-ES IV immunized mice. The K^b/IV Tet specifically stains CTL clones that recognize T ag epitope IV as well as T ag epitope IV-specific CTLs from rVV-ES IV immunized C57BL/6 mice.⁵ 501 siblings were identified in which one animal had a large osteosarcoma on the cervical vertebrae, and the other was judged tumor-free by X-ray. These mice were immunized at 11 months of age, along with 3-month-old 501 mice, and C57BL/6 mice of 3 and 12 months of age. All mice were sacrificed after 2 weeks, and their spleen cells were analyzed for the presence of CD8⁺, K^b/IV Tet⁺ cells. The number of CD8⁺ cells that could be induced to secrete IFN- after a short incubation with epitope IV synthetic peptide was determined with spleen cells from the same mice in parallel as a measure of function. C57BL/6 mice of both 3 and 12 months of age mounted robust epitope IV-specific responses in which 14–16% of CD8⁺ cells were stained by K^b/IV Tet (Fig. 9 A). In contrast, only 3.5% of CD8⁺ cells were K^b/IV Tet⁺ in 3-month-old 501 mice, indicating that fewer epitope IV-specific CD8⁺ cells develop in 3-month-old 501 versus C57BL/6 mice after immunization with rVV-ES IV. This is in contrast to the similar levels of lysis detected in bulk CTL cultures after in vitro restimulation (Fig. 4 , compare F and N). Staining of spleen cells from an 11-month-old tumor-free 501 mouse immunized with rVV-ES IV revealed a decrease in K^b/IV Tet⁺ cells to 0.5% of CD8⁺ cells (Fig. 9A ), suggesting that although bulk CTL responses against epitope IV are still detected in older 501 mice, the number of CD8⁺ cells that can respond diminishes with age. No such decrease was noted in C57BL/6 mice, indicating that decreased epitope IV responsiveness was attributable to endogenous T ag expression. Analysis of spleen cells from the tumor-bearing 501 sibling resulted in the inability to detect CD8⁺, K^b/IV Tet⁺ cells above background. This absence of epitope IV-specific CD8⁺ cells corresponds with the inability to expand epitope IV-specific CTL in vitro from tumor-bearing 501 mice. Analysis of naive spleen cells from either a 4-month-old 501 mouse or a tumor-bearing 10-month-old 501 mouse revealed that CD8⁺, K^b/IV Tet⁺ cells could not be detected above background levels (data not shown), indicating that immunization with rVV-ES IV was required to induce detectable levels of epitope IV-specific CD8⁺ T cells.

View larger version (40K): [in this window] [in a new window]   Fig. 9. Ex vivo detection of epitope IV-specific CD8+ T cells from 501 mice. 501 and C57BL/6 mice of the indicated ages were immunized i.v. with 1 x 107 pfu rVV-ES IV. After 2 weeks, spleen cells were harvested and stained with anti-CD8 plus H2-Kb/epitope IV tetramers (A) or for intracellular IFN- (B). Double-positive cells are indicated by the rectangular (A) or elliptical (B) regions. The percentage of CD8+ cells that are positive for H2-Kb/epitope IV or IFN- are indicated in each panel.

	DISCUSSION

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Our results demonstrate that the onset of tolerance to multiple CTL epitopes within SV40 T ag occurs with varied kinetics in line 501 mice. CTLs specific for the H2-D^b-restricted T ag epitope II/III were not detected in 3–4-month-old 501 mice using immunization with either T ag-transformed cells or rVVs expressing full-length T ag or epitope II/III as a minigene, suggesting that epitope II/III-specific CTLs are absent from the repertoire of 501 mice or that the onset of peripheral tolerance to epitope II/III occurs prior to 3–4 months of age. The low level CTL responses observed in 4-month-old 501 mice after immunization with full-length T ag can be partially overcome by immunization with rVVs expressing T ag epitopes I, IV, and V as minigenes, suggesting that more efficient delivery of the T ag epitopes into the class I MHC antigen presentation pathway enhances the ability to recruit T ag epitope-specific CTLs. As these 501 mice age and develop tumors, they fail to respond to T ag epitopes I, IV, and V, but the onset of tolerance to each epitope varies. Thus, distinct windows exist when CTLs can be activated to respond to T ag epitopes I, IV, and V during the life span of 501 mice. CTL tolerance to epitope I occurred by 6 months of age and corresponded with the time when maximum levels of T ag can be detected in the salivary glands. CTL responsiveness to epitope IV correlated with the appearance of primary osteosarcomas, because all tumor-bearing 501 mice tested failed to develop epitope IV-specific CTLs after immunization with rVV-ES IV. A third scenario was observed for the loss of CTL responses to the immunorecessive epitope V, in which tolerance correlated neither with the increased expression of T ag in the salivary glands or the progression of tumors but was apparent only in mice 12 months of age or older. These results indicate that a hierarchy of tolerance is established in 501 mice, which eliminates potentially tumor-reactive CTLs from the periphery.

Previous investigations of peripheral tolerance to SV40 T ag have used transgenic mice that express T ag from a tissue-specific promoter, such as the insulin promoter (3) . Such mice are responsive to T ag CTL epitopes in the absence of T ag expression in the thymus (8 , 11 , 14) , and activation of T ag-specific CTLs in these mice leads to the development of autoimmunity (11 , 13 , 15) . The fate of autoreactive CTLs in these mice, however, differs according to the model used. For example, mice that are transgenic for both full-length T ag and a TCR specific for an H2-K^k-restricted T ag CTL epitope developed spontaneous autoimmunity and failed to show tolerance to T ag (13 , 14) . In contrast, mice with fewer T ag-reactive CTLs showed signs of antigen specific tolerance, similar to our observations, which correlates with the onset of T ag expression in the periphery (11 , 13 , 16 , 17) . Using RIP1-Tag4 mice, which express T ag from the insulin promoter, Ye et al. (11) demonstrated that immunization of RIP1-Tag4 mice with SV40 induces T ag-specific CTLs and leads to a delay in the progression of T ag-induced tumors if mice are immunized prior to the onset of endogenous T ag expression. Immunization after the onset of T ag expression in the periphery failed to control tumor progression, suggesting that expression of the transgene leads to immune tolerance and that a period of time exists prior to T ag expression in the periphery when immunization leads to effective control of tumor progression. The present study, using 501 transgenic mice, reveals that the onset of CTL tolerance to multiple T ag epitopes can occur sequentially after expression of the transgene in the periphery, suggesting that distinct windows of opportunity exist for immunization against the T ag epitopes.

Although the specific immunological mechanism that leads to the onset of tolerance to T ag CTL epitopes in 501 mice remains to be determined, some possible mechanisms for the induction of tolerance by peripherally expressed tumor antigens include the induction of anergy, deletion of reactive CTLs, or suppression of immunity (40) . The induction of T cell anergy by peripherally expressed antigens has been reported for both CD4⁺ (41) and CD8⁺ (42) T cells and may be explained by the recognition of peptide ligands by naive T cells in the absence of costimulation from APCs (43) . Recognition of T-cell epitopes on tumor cells that lack the costimulatory molecules B7-1 (CD80) or B7-2 (CD86) lead to the induction of anergy (44 , 45) , suggesting that their interaction with CD28 on T cells is critical for the induction of tumor-specific immunity. Thus, direct recognition of T ag epitopes on peripheral tissues in 501 mice in the absence of proper costimulation could lead to the induction of CTL anergy in vivo.

Alternatively, naive T lymphocytes might be exposed to T ag epitopes on professional APCs in 501 mice through the mechanism of cross-presentation (46) . Several recent findings support an active role for professional APCs in the induction of T-cell tolerance. For example, engagement of the inhibitory receptor CTLA-4 on T lymphocytes by B7 molecules of activated APCs can induce tolerance directly, suggesting that antigen is transferred to APCs prior to tolerance induction (47) . Multiple studies have now determined that the induction of tolerance among naive T cells is preceded by a period of T-cell proliferation, which is thought to require interaction with professional APCs (41 , 48, 49, 50) . In addition, it is generally believed that naive lymphocytes of the adult do not traffic into peripheral nonlymphoid tissues, where they might be exposed to direct presentation of self epitopes (51 , 52) . Deletion of antigen-specific T cells after cross-presentation of peripherally expressed antigens by bone marrow-derived APCs has been observed for both CD8⁺ (49 , 53) and CD4⁺ (54) cells. Thus, the ability of professional APCs to process and present T ag after its expression in the periphery of 501 mice could explain the induction of tolerance to T ag CTL epitopes through cross-presentation. Whether the mechanism of tolerance induction involves deletion of epitope-specific CTL precursors or the induction of anergy remains to be determined.

Recent observations have indicated that the level of antigen expression in the periphery is critical for the induction of tolerance by cross-presentation (55 , 56) . Antigens expressed at low levels in the periphery either were ignored or induced weak proliferative responses in vivo and failed to induce deletion of autoreactive CD8⁺ T cells. Higher amounts of peripheral antigen, however, resulted in increased proliferation, followed by deletion of autoreactive CD8⁺ cells. These results indicate that the production of relatively small amounts of antigen in the periphery may not lead to efficient cross-presentation and manifests itself as immunological ignorance. In contrast, higher amounts of antigen production may lead to release of antigen that can be cross-presented by APCs and induce proliferation and subsequent deletion of autoreactive T cells. Thus, changes in the amount of T ag expressed during the life span of 501 mice might lead to the onset of tolerance to T ag CTL epitopes at distinct times.

At least two events in 501 mice might lead to increased levels of T ag in the periphery: (a) increased levels of T ag can be detected in the salivary glands of 501 mice at 6 months of age.⁴ This time of increased antigen load corresponds with the loss of reactivity to T ag epitope I. Although no direct correlation was established between these two events, our results might be explained by an increase in the availability of H2-D^b/epitope I complexes in the periphery, which lead to the induction of tolerance among epitope I-specific CTL precursors; and (b) the progression of T ag-expressing tumors in 501 mice might lead to the release of increased amounts of T ag, particularly as the tumors undergo necrosis or apoptosis. Thus, this second wave of T ag expression might result in increased exposure of epitope IV-specific CTLs to cross-presented antigen. Our results using K^b/IV tetramers indicate that many of the epitope IV-specific CTLs are lost prior to the detection of osteosarcomas, because fewer epitope IV-specific CTLs were detected in 12-month-old, tumor-free 501 mice than in 4-month-old, tumor-free 501 mice. This suggests that tolerance to epitope IV progresses over the life span of 501 mice and is accelerated by the appearance of tumors. Importantly, T ag expression was maintained in the tumors of 501 mice that developed tolerance to epitope IV. These findings are reminiscent of experiments by Kurts et al. (49) in which a steady decline in the number of OVA-specific TCR transgenic T cells was observed after transfer into OVA transgenic mice (RIP-mOVA). Thus, our data support a model in which the pool of T ag epitope-specific CTLs are depleted over time because of increased levels of the transgene in the periphery, finally resulting in the complete loss of T ag epitope-specific CTLs.

Several possible explanations can be offered regarding the different times for the onset of tolerance to T ag epitopes I, IV, and V. As indicated above, the amount of antigen expressed in the periphery appears to be a determining factor in the onset of CTL tolerance (55 , 56) . Although previous studies examined only the response to a single CTL epitope, we suggest that the levels of individual T ag epitopes presented for CTL recognition in the periphery of 501 mice may vary, subject to the binding properties of the epitopes themselves. We have shown previously that T ag epitope I forms extremely stable complexes with H2-D^b molecules (28) . This could lead to the accumulation of a large number of D^b/epitope I complexes at the surface of T ag-expressing cells or APCs processing T ag, which might lead to the relatively early onset of tolerance to epitope I. In contrast, T ag epitope V dissociates rapidly from H2-D^b and is not expected to accumulate at the cell surface. Thus, the frequency with which epitope V-specific CTL precursors encounter D^b/epitope V complexes in 501 mice might be low. T ag epitope IV forms complexes with H2-K^b having an intermediate stability and, therefore, might accumulate an intermediate number of complexes at the cell surface. Thus, varying numbers of T ag epitope/MHC class I complexes may contribute to the development of the hierarchy of tolerance to T ag epitopes observed in 501 mice.

Although immunization with full-length T ag results in the simultaneous induction of CTLs specific for epitopes I and IV, we have shown previously that the frequency of epitope IV-specific CTLs is 4–5-fold higher than epitope I-specific CTLs in C57BL/6 mice using both limiting dilution analysis (30) and tetramer staining of ex vivo lymphocytes.⁵ Thus, another explanation for the more rapid onset of tolerance to epitope I than to epitope IV in 501 mice is that fewer epitope I-specific CTL precursors than epitope IV-specific CTL precursors need to be affected to result in loss of CTL responsiveness. In support of this scenario, Morgan et al. (56) have shown that the time required to develop peripheral tolerance against a HA CTL epitope in InsHA transgenic mice was directly related to the number of HA-specific TCR transgenic CD8⁺ cells that were injected. An increase in the number of HA-specific CD8⁺ T cells transferred into InsHA mice resulted in an increase in the amount of time required to develop peripheral tolerance to HA. Thus, the starting frequency of epitope-specific CTLs in 501 mice, as well as the level of epitope presentation in the periphery might contribute to the hierarchy of tolerance onset observed in our experiments. We are currently addressing these possibilities.

Our results indicate that the ability to develop CTLs against epitope I in 501 mice was dependent on CD4⁺ T cells, because 4-month-old 501/CD4^-/- mice failed to develop epitope I-specific CTLs after immunization with rVV-ES I but were capable of responding to epitopes IV and V after immunization with rVV-ES IV or rVV-ES V, respectively. In contrast, CD4⁺ cells were not required in T ag-negative animals to develop epitope I-specific CTLs. Thus, epitope I-specific CTL precursors are either absent from the T-cell repertoire of 501/CD4^-/- mice or fail to become activated in the absence of CD4⁺ T-cell help in these mice. Studies on the role of CD4⁺ T cells in the generation of CTL responses using various systems have revealed that some CTL responses were undetectable (57 , 58) , whereas others apparently were unaffected (59 , 60) by the absence of CD4⁺ cells. More recent studies, however, have shown that although CTLs are generated in the absence of CD4⁺ T cells, optimal CTL responses require CD4⁺ T-cell help (61, 62, 63, 64) . Thus, the generation of epitope I-specific CTLs in 501 mice might be more dependent on CD4⁺ T cells than in C57BL/6 mice, attributable to tolerogenic mechanisms that reduce the level of CTL responsiveness in 501 mice. The role of CD4⁺ T-cell help in promoting effective CTL responses recently has been attributed to their ability to "condition" APCs for the subsequent stimulation of CTLs (65, 66, 67) and involves the delivery of a signal through CD40-CD40 ligand.

In addition, the presence of CD4⁺ T cells may moderate the onset of tolerance in 501 mice. The addition of OVA-specific TCR transgenic CD4⁺ T cells was shown to prevent the deletion of OVA-specific TCR transgenic CD8⁺ T cells by the cross-presentation of peripheral OVA in RIP-mOVA transgenic mice (68) , suggesting that CD4⁺ cells were required for survival of autoreactive CTLs in this model system. Thus, in the absence of CD4⁺ T-cell help, epitope I specific CTL may be deleted more rapidly in 501 transgenic mice. In support of this idea, we have obtained preliminary evidence that the onset of tolerance to epitopes IV and V is also accelerated in 501/CD4 knockout mice compared with 501 mice (data not shown).

We have shown that an efficient CTL response against three of four T ag epitopes can be induced after immunization of 501 mice with T ag epitope minigenes and that the onset of CTL tolerance is epitope specific. Whether the induction of CTLs against one or more of the T ag epitopes prior to the onset of tolerance in 501 mice will have an effect on tumor progression remains to be determined. These findings, however, indicate that distinct windows of opportunity exist to activate epitope-specific CTLs against endogenous T ag-induced tumors in 501 mice. Thus, in the tumor-bearing host, the effectiveness of immunization against a particular tumor antigen CTL epitope might be determined by the timing of tolerance onset.

	ACKNOWLEDGMENTS

 
We sincerely thank Drs. Alina Boesteanu and Sebastian Joyce for assistance in the preparation of tetramers. We also thank Dr. James M. Griffith for detailed pathological descriptions of osteosarcomas and Dr. Ronald P. Wilson for assistance with X-ray examination of mice. We thank Melanie Epler and Andrew Gaydos for excellent technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Research Grants CA25000 (to S. S. T.) and CA37102 (to B. B. K.) from the NIH and CORE Grant P30 CA34196 from the National Cancer Institute (to B. B. K.). T. D. S. is supported by the Concern Foundation for Cancer Research/Cancer Research Institute Fellowship.

² To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, H107, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8872; Fax; (717) 531-5578; E-mail: sst1{at}psu.edu

³ The abbreviations used are: T ag, T antigen; FBS, fetal bovine serum; rVV, recombinant vaccinia virus; APC, antigen presenting cell; ES, adenovirus E3/19K endoplasmic reticulum insertion sequence; HA, hemagglutinin; OVA, ovalbumin; pfu, plaque forming unit(s); TCR, T-cell receptor; HSV, herpes simplex virus.

⁴ I. Marton, S. E. Johnson, I. Damjanov, K. S. Currier, J. P. Sundberg, and B. B. Knowles. Expression and immune recognition of SV40 Tag in transgenic mice that develop metastatic osteosarcomas, submitted for publication.

⁵ L. M. Mylin, T. D. Schell, M. Epler, D. Roberts, A. Bosteanu, E. J., Collins, J. A. Frelinger, S. Joyce, and S. S. Tevethia. Quantitation of CD8+ T lymphocyte responses to multiple epitopes from SV40 large T antigen in C57BL/6 mice immunized with SV40, SV40 T antigen-transformed cells, or with vaccinia virus recombinants expressing full-length T antigen or epitope minigenes, submitted for publication.

Received 10/21/99. Accepted 3/29/00.

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