HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Staveley-O’Carroll, K. Articles by Tevethia, S. S. Search for Related Content PubMed PubMed Citation Articles by Staveley-O’Carroll, K. Articles by Tevethia, S. S.

In Vivo Ligation of CD40 Enhances Priming Against the Endogenous Tumor Antigen and Promotes CD8⁺ T Cell Effector Function in SV40 T Antigen Transgenic Mice¹

Kevin Staveley-O’Carroll^*,, Todd D. Schell^*, Marcela Jimenez, Lawrence M. Mylin^2,*, M. Judith Tevethia^*, Stephen P. Schoenberger and Satvir S. Tevethia^3,*

Departments of ^* Microbiology and Immunology and Surgery, Pennsylvania State University College of Medicine, Hershey, PA 17033; and Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability to initiate and sustain CD8⁺ T cell responses to tumors in vivo is hindered by the development of peripheral T cell tolerance against tumor-associated Ags. Approaches that counter the onset of T cell tolerance may preserve a pool of potentially tumor-reactive CD8⁺ T cells. Administration of agonist Ab to the CD40 molecule, expressed on APCs, can enhance immunization approaches targeting T lymphocytes in an otherwise tolerance-prone environment. In this report, the effects of anti-CD40 administration on priming of naive CD8⁺ T cells against an endogenous tumor Ag were investigated. Line 501 mice express the SV40 large T Ag oncoprotein as a transgene from the -amylase promoter, resulting in the development of peripheral CD8⁺ T cell tolerance to the H-2-D^b-restricted immunodominant epitope I of T Ag by 6 mo of age, before the appearance of osteosarcomas. We demonstrate that naive epitope I-specific TCR transgenic (TCR-I) T cells undergo peripheral tolerance following adoptive transfer into 6-mo-old 501 mice. In contrast, administration of agonistic anti-CD40 Ab led to increased expansion of TCR-I T cells in 501 mice, the acquisition of effector function by TCR-I T cells and the establishment of T cell memory. Importantly, this enhanced priming effect of anti-CD40 administration did not require immunization and was effective even if administered after naive TCR-I T cells had encountered the endogenous T Ag. Thus, anti-CD40 administration can block the onset of peripheral tolerance and enhance the recruitment of functionally competent effector T cells toward an endogenous tumor Ag.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Due to their specificity and potent effector function, CD8⁺ T cells are an attractive target population for immunotherapeutic approaches to cancer. Many tumor-associated Ags recognized by CD8⁺ T cells have now been identified and represent nonmutated self-Ags that can serve as the focus for vaccination approaches to cancer (1, 2). Several mechanisms have been identified, however, that might limit the effectiveness of targeting self-reactive T cells, including deficiencies in the T cell repertoire due to negative selection of potentially tumor Ag-reactive T cells (3, 4, 5, 6) as well as regulatory mechanisms that down-modulate immune responses to self-Ags in the periphery (7). In particular, recent evidence suggests that the recognition of peripheral self-Ags by naive CD8⁺ T cells leads to activation, expansion, and subsequent deletion of these self-reactive T cells to maintain tolerance to self-Ags in the periphery (8). Thus, strategies that protect naive tumor-reactive T cells from destruction or lead to the expansion and maintenance of effector and memory T cells within the tumor-bearing host will be critical for designing immunotherapeutic approaches to cancer.

Mice that develop spontaneous tumors due to the transgenic expression of oncogenes provide realistic models to assess immunotherapeutic strategies for potential use in the treatment of human cancer. Expression of the large tumor Ag (T Ag)⁴ from SV40 as a transgene in mice leads to the development of spontaneous tumors (9, 10). This oncogenic activity stems from the ability of the T Ag to inactivate the tumor suppressor proteins Rb and p53 as well as initiate cell cycle progression (11). In addition, T Ag serves as the target of a strong CTL response that can lead to the rejection of T Ag expressing tumors (12). Immunization of C57BL/6 mice with wild-type T Ag leads to the development of CTL specific for these three immunodominant epitopes: designated epitope I (T Ag residues 206–215), epitope II/III (T Ag residues 223–231), and epitope IV (T Ag residues 404–411) (13, 14, 15). Epitopes I and II/III are H-2-D^b restricted, whereas epitope IV is H-2-K^b restricted.

Expression of T Ag as a transgene also can lead to the onset of either central or peripheral CD8⁺ T cell tolerance to the T Ag epitopes, depending on the site and timing of transgene expression (4, 16, 17, 18, 19). Line B6/501 (H-2^b) transgenic mice express T Ag under the -amylase promoter, resulting in the expression of T Ag in the salivary glands and in bone (20, 21). Although neoplasia is not detected in the salivary glands, T Ag expression in bone leads to the development of osteosarcomas that can metastasize to the liver and lung. The expression of T Ag as a self Ag in 501 mice leads to the progressive loss of CTL responses to the T Ag epitopes (19). In particular, loss of CTL responsiveness to T Ag epitope I is tightly associated with the onset of T Ag expression in 501 mice. Immunization of 501 mice at 6 mo of age, when T Ag is expressed at high levels in the salivary glands (21), failed to induce epitope I-specific CTL, although immunization of these mice at 3–4 mo of age resulted in the successful recruitment of epitope I-specific CTL (19). Whether the loss of epitope I-specific CTL activity in line 501 mice is due to the peripheral deletion of reactive CD8⁺ T cells or the induction of unresponsiveness is unknown.

The presence of potentially autoreactive cells in peripheral lymphoid organs can result in either ignorance of cognate Ag, unless a response is initiated by exogenous immunization (22, 23, 24), or Ag encounter resulting either in an immune response (25, 26) or the induction of tolerance (19, 27, 28, 29, 30). It has become increasingly clear that the nature of the immune response to peripheral Ag is largely determined by the characteristics of the APC (31). Both T cell activation and the induction of peripheral T cell tolerance can be mediated through recognition of Ag on bone marrow-derived APCs, which are most likely dendritric cells (DCs) (28, 32, 33, 34). Recent evidence suggests that tissue-resident DCs with an immature phenotype acquire Ag from the surrounding tissues during noninflammatory conditions (35) and migrate to secondary lymphoid organs where an encounter with Ag-specific T cells can result in tolerance to these self-Ags. The amount of self-Ag expressed in the periphery may be one factor that determines whether a naive T cell encounter with APCs results in tolerance or ignorance, as recent studies indicate that high levels of endogenous Ag can lead to the induction of T cell tolerance (36, 37).

Recent approaches to immunotherapy have focused on modifying the maturation status of APCs such that T cell encounter with APCs leads to full activation of naive T lymphocytes. One promising approach has been the engagement of CD40 on the APC via soluble CD40 ligand or agonist anti-CD40 Abs. CD40 on APCs is naturally engaged by the ligand CD154 (CD40 ligand) on activated CD4⁺ T cells, resulting in APC activation and differentiation (38). Ligation of CD40 leads to changes in the APC phenotype, including increased surface expression of MHC molecules and costimulatory receptors and the production of high levels of the T cell-stimulating cytokine IL-12 (39, 40, 41). In addition, CD40 ligation induces the migration of APCs to the secondary lymphoid tissue (42) where they can engage naive T cells. Ligation of CD40 in vivo following the injection of agonistic anti-CD40 Abs can substitute for CD4⁺ T cell help in the priming of naive CD8⁺ T cells, suggesting that T cell help may be mediated primarily via CD40 ligand-CD40 interactions with APCs (43, 44, 45). Several recent reports have demonstrated that the injection of agonistic anti-CD40 Abs can prevent or delay the onset of peripheral T cell tolerance (46, 47, 48). In addition, injection of mice with anti-CD40 in combination with specific immunization can lead to enhanced immunity to transplantable tumors (46, 47, 49, 50) or spontaneous tumors expressing a neo-Ag (51). Thus, anti-CD40 ligation may result in both the preservation of the host self-reactive T cell repertoire and enhanced expansion of tumor-reactive T cells in vivo.

In this report, the fate of naive T Ag epitope I-specific TCR transgenic (TCR-I) CD8⁺ T cells was determined after exposure to the endogenous T Ag in 501 T Ag transgenic mice. The results indicate that epitope I-specific CD8⁺ T cells proliferated and developed an activated phenotype in lymph nodes (LNs) draining areas of T Ag expression; however, these cells failed to acquire effector function. In vivo treatment with anti-CD40 Ab resulted in enhanced expansion of epitope I-specific T cells and promoted the acquisition of effector function in the absence of exogenous immunization. Importantly, this effect was observed even if anti-CD40 treatment was administered after exposure of the epitope I-specific T cells to the endogenous T Ag and led to long-term maintenance of epitope I-specific CD8⁺ T cells. Thus, in vivo CD40 ligation results in preservation and expansion of tumor-reactive and functional effector T cells that might be targeted for subsequent immunotherapy of tumors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Male C57BL/6 (H-2^b) mice (4–6-wk-old) were purchased from The Jackson Laboratory (Bar Harbor, ME), maintained in isolator cubicles at the animal facility of the Milton S. Hershey Medical Center, Hershey, PA) and routinely used between the ages of 7 and 12 wk. Line C57BL/6-TgN (Amy1Tag) 501Knw, or 501, mice express full-length SV40 T Ag as a transgene from the -amylase promoter and have been previously described (20, 21). The 501 mice were bred and maintained at the animal research facility of the Milton S. Hershey Medical Center. B6.SJL mice were purchased from Taconic Farms (Germantown, NY) and bred and maintained at the animal research facility of the Milton S. Hershey Medical Center. All animal studies were performed in accordance with guidelines established by the Pennsylvania State University College of Medicine Animal Care and Use Committee under an approved protocol.

Cloning of the epitope I-specific TCR from CTL clone Y-1

TCR sequences for both the - and -chains were derived from the epitope I-specific CTL clone Y-1 (52). To determine the nucleotide sequence of the combining regions for each subunit, total RNA was extracted from clone Y-1 cells using TriReagent (Sigma-Aldrich, St. Louis, MO) and reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) utilizing an oligo(dT) reverse primer. The TCR -chain combining region was amplified by PCR utilizing an antisense 3' constant region oligonucleotide (5'-CGAGGATCTTTTAACTGGTA-3'; Ref.53) and a 5' variable region V3-chain sense oligonucleotide (5'-TCCTTCCACCTGCGGAAAGCC-3'; Ref.53) while the TCR V-chain combining region was amplified using the 3' antisense constant region oligonucleotide (5'-CTTGGGTGGAGTCACATTTCT-3'; Ref.54) and a 5' sense V7 oligonucleotide (5'-AAGAAGCGGGAGCATTTCTTC-3'; Ref.55). The TCR and TCR PCR products were gel isolated, phosphorylated using T4 polynucleotide kinase (Life Technologies, Grand Island, NY), and rendered blunt-ended with T4 DNA polymerase and subcloned into pUC19. Sequence analysis of the TCR -chain sequence revealed J region 1.4 use while analysis of the TCR sequence revealed J41 use. Accordingly, 3' antisense genomic primers corresponding to intron sequences flanking the respective TCR and TCR J regions (J41, 5'-TGCGGCCGCTTTTTTTTTACTTACTTGGAATGACAGTC-3'; Ref.56); J1.4, 5'-AGGAGTTCCCCGCGGCTCTAGGTTTACAAC-3'; Ref.54) were used in combination, respectively, with 5' sense primers corresponding to 5' noncoding sequences of V3.1 (5'-GTTCCCGGGTTCCTCCACAACAGAGCTGCAGCCT-3'; Ref.53) or V7 (5'-CACACTTTCCTCGAGACCACCATGAGAGTTAGG-3'; Ref.55) to amplify the genomic sequences from Y-1 cell-derived DNA and incorporate restriction endonuclease cleavage sites at the ends of each product (, 5'XmaI, 3'NotI; , 5'XhoI, 3'KspI/SacII). The amplified genomic V(D)J fragments were subcloned into PUC19 as blunt-ended fragments, the nucleotide sequences were verified, and the fragments were excised by restriction digestion and ligated into the appropriately digested TCR - or -expression cassette plasmids (pTcass and pTcass, respectively; obtained from Dr. D. Mathis, Institute de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France; Ref.57). Recombinant cassette vectors containing the appropriate Y-1 TCR - and -chain fragments were identified by restriction digestion analysis, and the presence of the proper V(D)J inserts verified by PCR amplification. Plasmids were purified by cesium chloride gradient centrifugation and digested (, SalI; , KpnI) to liberate a fragment bearing the respective subunit expression cassettes, which were isolated from agarose gels using the QIAEXII method (Qiagen, Valencia, CA) and eluted directly into microinjection buffer. Fragment solutions were stored at -80°C.

Generation of T Ag TCR-I mice

Purified Y-1 TCR - and -chain expression cassettes were combined before injection. Microinjection of fertilized embryos from B6D2F₁/J mice was performed as previously described (58). The presence of the and transgene(s) in weanlings was determined at 4 wk of age by PCR analysis of tail-derived DNA using the following primer pairs: V3.1 chain sense, 5'-TCCTTCCACCTGCGGAAAGCC-3'; J41 chain antisense, 5'-TGCGGCCGCTTTTTTTTTACTTACTTGGAATGACAGTC-3'; V7 chain sense, 5'-AAGAAGCGGGAGCATTTCTCC-3'; and J1.4 chain antisense, 5'-AGGAGTTCCCCGCGGCTCTAGGTTTACAAC-3'. Amplification of the corresponding 180- and 200-bp fragments from genomic DNA was diagnostic for the presence of the transgenes. Amplification of a 450-bp segment of the p53 gene using previously described primers served as a control for the integrity of the DNA (4, 59). Expression of the transgene products was confirmed by staining lymphocytes from various lymphoid tissues with TCRV7-specific mAb (BD PharMingen, San Diego, CA), anti-TCRV3.1 antisera (generously provided by Drs. B.-C. Sim and N. Gascoigne, The Scripps Research Institute, La Jolla, CA) (60) and Db/epitope I tetramer (61). Two founder lines were derived and designated as lines 416 and 422. Line 416 mice were maintained by backcrossing transgene-positive males with C57BL/6 females followed by screening for the presence of the transgene by PCR analysis as described below. Line 416 mice have been backcrossed to C57BL/6 mice for over 10 generations.

Cell lines and media

B6/K-TagI cells were derived by transformation of C57BL/6 primary kidney cells with plasmid pLM506-G(DC-1), which encodes a T Ag variant containing alanine substitutions of residues N227, F408, and N493. Plasmid pLM506-G(DC-1) was constructed by the Altered Sites oligonucleotide-directed mutagenesis procedure (Promega) using as a template ssDNA derived from pSelect-ESV (14, 61). Because residues N227, F408, and N493 constitute critical anchor residues within epitopes II/III, IV, and V, respectively, alanine substitution effectively abrogates both the immunogenicity and antigenicity of epitopes II/III, IV, and V (data not shown). Cell lines were maintained in DMEM supplemented with 100 U penicillin/ml, 100 µg streptomycin/ml, 100 µg kanamycin/ml, 2 mM L-glutamine, 10 mM HEPES buffer, 0.075% (w/v) NaHCO₃, and 5% FBS. All lymphocyte manipulations were prepared using complete RPMI 1640 medium, which contained 100 U penicillin/ml, 100 µg streptomycin/ml, 2 mM L-glutamine, 10 mM HEPES buffer, 50 µM 2-ME, and 10% FBS.

Synthetic peptides and viruses

Peptides 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; Millipore, Bedford, MA). Peptides were solubilized in DMSO and diluted to the appropriate concentration with RPMI 1640 medium. Peptides used in these experiments correspond to SV40 T Ag epitopes I (SAINNYAQKL) and influenza virus (Flu) nucleoprotein (NP) 366–374 (ASNENMETM). The recombinant vaccinia virus (rVV) encoding T Ag epitope I as a minigene, rVV-I, has been previously described (28).

Flow cytometric analysis of lymphocyte populations by surface and intracellular cytokine staining

Ex vivo staining of lymphocytes was performed on single-cell suspensions obtained from cervical LNs and spleens, as previously described (61), that had been depleted of RBCs by centrifugation over a Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) gradient according to the manufacturer’s specifications. Production and characterization of the H-2-D^b/T Ag epitope I (Db/I) and H-2-D^b/influenza virus NP epitope 366–374 (Db/Flu) tetramers (Tet) and surface staining of lymphocytes with MHC tetramers and primary conjugated Abs was performed as previously described (61). The following Abs were purchased from BD PharMingen: FITC-, PE-, or Cychrome-labeled anti-CD8a (clone 53-6-7), PE-labeled mouse anti-mouse CD45.1 (clone A20), FITC-labeled rat anti-mouse CD44 (clone IM7), FITC-labeled hamster anti-mouse CD69 (clone H1.2F3), FITC-labeled rat anti-mouse TCRV7 (clone TR310), unlabeled rat anti-mouse CD16/CD32, and FITC-labeled rat anti-mouse IFN- (clone XMG1.2). The rabbit anti-mouse V3.1 antisera (60) was used in conjunction with a secondary goat anti-rabbit-FITC conjugate (BD PharMingen). Cells were washed, fixed with 2% paraformaldehyde in PBS, and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed and prepared using CellQuest software (BD Biosciences) or FlowJo software (Tree Star, San Carlos, CA). In some cases, the percentage of CD8⁺ cells that stained specifically with Db/I Tet was determined by subtracting the percentage of CD8⁺ Db/Flu Tet⁺ cells from the percentage of CD8⁺ Db/I Tet⁺ cells within the same population. Intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s specifications and as previously described (61). Where indicated, the percentage of CD8⁺ cells that express intracellular IFN- was calculated by subtracting the percentage of cells that produce IFN- following exposure to an unrelated peptide from those that stained for IFN- after stimulation with T Ag epitope I peptide.

Adoptive transfers, anti-CD40 treatment, and immunizations

Six-month-old C57BL/6 and 501 mice received adoptive transfers with RBC-depleted lymphocytes derived from spleens and LNs of line 416 TCR-I mice. Recipients were injected i.v. in the tail vein with lymphocytes derived from line 416 mice containing 5 x 10⁶ clonotypic TCR-I CD8⁺ T cells, as determined by staining with Db/epitope I tetramer, in 200 µl HBSS. Mice received 100 µg of purified agonist anti-CD40 Ab FGK45 (62) or polyclonal rat IgG (Sigma-Aldrich) by i.p. injection at the indicated times. Some mice were injected i.p. with 3 x 10⁶ B6/K-TagI cells or with 1 x 10⁷ PFU of the indicated rVV.

In vivo proliferation assay

For in vivo proliferation assays (63, 64), RBC-depleted spleen cells from line 416 mice were resuspended at 1 x 10⁷/ml in PBS/0.1% BSA and labeled with 5 µM 5- and 6-CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. Cells were then washed three times in PBS, resuspended in HBSS, and injected i.v at a dose of 5 x 10⁶ clonotypic TCR-I T cells per mouse. After 8 days, spleens and LNs from recipient mice were harvested, and the intensity of CFSE staining was measured among CD8⁺ Db/I Tet⁺ cells.

In vivo cytotoxicity assay

Six-month-old 501 and C57BL/6 mice were injected with 5 x 10⁶ clonotypic TCR-I T cells. After 14 days, RBC-depleted spleen cells from B6/SJL congenic mice were pulsed with either 1 µM T Ag epitope I peptide or the control Flu NP 366–374 peptide for 90 min at 37°C, 5% CO_2. Excess peptide was removed with five washes and cells were resuspended at 1 x 10⁷ cells/ml in PBS/0.1% BSA. Epitope I-pulsed cells were labeled with 5 µM CFSE (CFSE^high cells) and Flu NP peptide-pulsed cells were labeled with 0.5 µM CSFE (CFSE^low cells) for 10 min at 37°C. Labeling reactions were terminated by the addition of ice-cold PBS followed by two washes in PBS. Mice received an i.v. injection containing a mixture of 2.5 x 10⁶ CFSE^low cells and 2.5 x 10⁶ CFSE^high cells in 200 µl HBSS. After 6 h, spleens from recipient mice were harvested, and CFSE^high CD45.1⁺ and CFSE^low CD45.1⁺ cells were quantitated by flow cytometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endogenous T Ag epitope I-specific CD8⁺ T cells are undetectable in 6-mo-old 501 mice

We have shown previously that 3-mo-old 501 mice immunized with a rVV-encoding epitope I as a minigene induces CTL specific for T Ag epitope I, whereas 6-mo-old 501 mice failed to develop detectable levels of epitope I-specific CTL, even after in vitro expansion (19). Because detection of epitope I-specific CTL required both expansion and the development of effector function, it remained to be determined whether the endogenous cells were deleted, failed to proliferate in response to immunization against epitope I, or proliferated but failed to acquire effector function. To examine whether a nonfunctional population of epitope I-specific CD8⁺ T cells accumulated in 6-mo-old 501 mice following immunization against epitope I, MHC class I tetramers (Db/I Tet) were utilized to detect the presence of epitope I-specific CD8⁺ T cells directly ex vivo. Nine days after vaccination with rVV-I, C57BL/6 mice developed epitope I-specific CD8⁺ T cells (1.5% of all CD8⁺ cells) detectable by ex vivo staining with Db/I Tet, while epitope I-specific CD8⁺ T cells were undetectable in 501 mice (Fig. 1). Thus, 501 mice failed to accumulate detectable numbers of endogenous epitope I-specific CD8⁺ T cells following specific immunization.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Line 501 mice are tolerant to T Ag epitope I. Six-month-old C57BL/6 or 501 mice were vaccinated with 1 x 107 PFU rVV-I. Nine days later, spleens were harvested and lymphocytes stained with anti-CD8a and either Db/epitope I tetramer (Db/I Tet) or a control tetramer constructed using the unrelated Db-restricted epitope NP 366–374 from influenza virus (Db/Flu Tet). The upper right quadrant represents epitope I-specific CD8+ T cells. The percentage of CD8+ T cells specific for epitope I is indicated.

To develop a system in which the fate of naive epitope I-specific CD8⁺ T cells could be monitored in 501 mice, a mouse line expressing the TCR - and -chains of the T Ag epitope I-specific CTL clone Y-1 was derived. (Fig. 2A). Two founder lines were identified and designated lines 416 and 422. Characterization of line 416 is described in this report. Expression of the transgene products in each line was detected by immunofluorescence staining with Db/I Tet, and Abs specific for the transgenic TCR V3.1 and V7 chains. Analysis of spleen cells from line 416 mice revealed that 94% of the CD8⁺ T cells were specific for T Ag epitope I (Fig. 2B), although 100% of the CD8⁺ T cells expressed TCRV7 (Fig. 2B). This discrepancy was explained by the finding that 87% of the TCRV7⁺ CD8⁺ T cells coexpressed the transgenic TCRV3.1 chain (Fig. 2B, left histogram), consistent with the percentage of CD8⁺ T cells that stained positive with the Db/I Tet. The specificity of the rabbit anti-V3.1 sera is indicated by the lack of positive staining on CD8⁺, TCRV7⁺ T cells in C57BL/6 spleen cells (Fig. 2B, left histogram). The CD8⁺ TCR-I T cells derived from line 416 mice were determined to be functional as in vitro stimulation of line 416 spleen cells with T Ag transformed cells induced epitope I-specific CTL (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Characterization of line 416 TCR-I transgenic mice. A, Predicted amino acid sequence for the combining regions of the TCR - and -chains derived from CTL clone Y-1 used to develop TCR transgenic line 416. B, Staining of spleen cells from control C57BL/6 or TCR-I transgenic line 416 mice using the indicated Abs or Db/I Tet. The gated regions in the lower right panel indicate the CD8+ Db/I Tet+ and CD8+, Db/I Tet- populations, and the corresponding histograms indicate the level of TCRV7 expression. The percentage of cells within each gate is indicated. The lower left histogram indicates the percentage of CD8+ TCRV7+ spleen cells that stain positively for TCRV3.1.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Activation and expansion of TCR-I T cells in 501 mice in the absence of effector function. A, Five million clonotypic TCR-I cells from line 416 TCR transgenic mice were transferred into 6-mo-old 501 or C57BL/6 mice. At 7 and 14 days after transfer, T cells isolated from the spleen and cervical LNs were stained with anti-CD8a and either a T Ag epitope I MHC tetramer (Db/I Tet) or a control tetramer (Db/Flu Tet). Results were quantitated by flow cytometric analysis. The data are representative of three mice per group and the experiment was repeated twice with similar results. The percentage of CD8+ T cells that stained positive with each tetramer is indicated in the upper right quadrant of each dot plot. B, A dose of 5 x 106 clonotypic TCR-I cells were transferred into 6-mo-old 501 or C57BL/6 mice. Seven days after transfer, cells were isolated from cervical LNs. Triple staining was performed using Db/epitope I tetramer, anti-CD8a, and Abs to either CD44 or CD69. Pairs of histograms (upper and lower) represent CD8+ T cells that either were (CD8+, Db/I Tet+) or were not (CD8+, Db/I Tet-) specific for T Ag epitope I. The percentage of CD8+ T cells within each population that had up-regulated CD44 or CD69 is indicated above the marker. C, A dose of 5 x 106 clonotypic TCR-I cells from line 416 mice were transferred into 501 or C57BL/6 mice. At 7 and 14 days after transfer, lymphocytes isolated from the draining cervical LNs and spleen were stimulated with 1 µM of either epitope I peptide or the control peptide NP 366–374 from influenza virus for 6 h in the presence of brefeldin A. CD8+ T cells were then stained for the production of intracellular IFN- and quantitated by flow cytometry. The percentage of CD8+ T cells producing IFN- is indicated in the upper right of each quadrant. The data shown are representative of three mice.

To determine whether Ag encounter by TCR-I T cells in 501 mice resulted in the acquisition of effector function, CD8⁺ T cells re-isolated from 501 mice were tested for their ability to produce IFN- in response to epitope I. Freshly isolated cervical LN or spleen cells were stimulated in vitro with epitope I peptide or a control peptide (Flu NP 366–374) in the presence of brefeldin A for 6 h and then stained for accumulation of intracellular IFN-. As expected, cells re-isolated from C57BL/6 mice that were never exposed to T Ag failed to produce IFN- in response to epitope I peptide (Fig. 3C). Lymphocytes isolated from 501 mice, which developed an Ag experienced phenotype in LNs draining sites of T Ag expression (Fig. 3B), also failed to produce significant amounts of IFN- (Fig. 3C). Although 8–9% of CD8⁺ T cells from the cervical LNs of 501 mice were positive by tetramer staining (Fig. 3A), <1% of the CD8⁺ T cells from these same LNs were capable of producing IFN- after stimulation with epitope I peptide. Thus, although TCR-I T cells accumulated in LNs draining the site of T Ag expression and developed an activated phenotype in 501 mice, they failed to acquire effector function.

TCR-I T cells exposed to endogenous T Ag respond inefficiently to subsequent immunization

To determine whether naive TCR-I T cells exposed to the endogenous T Ag in 501 mice are compromised in their ability to respond to T Ag epitope I in vivo, 6-mo-old 501 and control C57BL/6 mice were given adoptive transfers with 5 x 10⁶ clonotypic TCR-I T cells, rested for 2 wk and then immunized with rVV-I. Representative mice from each group were sacrificed at day 14, before immunization, to determine the frequency of TCR-I T cells present at this time point. TCR-I T cells were present at a frequency of 1–2% of CD8⁺ T cells in both the spleen and cervical LNs of C57BL/6 recipients (Fig. 4B). As shown previously (Fig. 3), while the frequency of TCR-I T cells present in the spleen of 501 mice at day 14 posttransfer was similar to that detected in C57BL/6 mice, increased numbers of TCR-I T cells accumulated in the cervical LNs of 501 mice at this time point (Fig. 4A).

View larger version (68K): [in this window] [in a new window]   FIGURE 4. Naive TCR-I T cells become tolerant following transfer into 501 mice. Six-month-old 501 and C57BL/6 (B6) mice were reconstituted with 5 x 106 clonotypic TCR-I T cells and rested until day 14 following adoptive transfer. Lymphocytes from spleen and LN of representative mice were analyzed at day 14 to determine the percentage of CD8+ T cells specific for T Ag epitope I by staining with MHC tetramers (mouse #1 and #2). Half of the remaining mice in each group were immunized with 1 x 107 PFU rVV-ES-I at day 16 postadoptive transfer and then sacrificed 9 days later (day 25 post-TCR-I transfer) for analysis of spleen and LN populations. The percentage of CD8+ T cells that stained positive with Db/I Tet is indicated in upper right of each histogram for both immunized (mouse #3 and #4) and unimmunized (mouse #5 and #6) mice. The data represent 100,000 events. *, Only 10,000 events were collected for B6#3 LN.

Expansion of effector TCR-I T cells following conditioning of 501 mice with activating anti-CD40 Ab

Professional APCs can be stimulated to increase their Ag-presenting activity and immunogenic potential by ligation of their CD40 receptor (39, 45, 65). This is mediated through engagement of CD154, a TNF family member expressed on activated CD4⁺ T cells, platelets, and mast cells (38). The engagement of CD40 by CD154 can be replaced by the administration of the FGK45 agonistic Ab to CD40, bypassing the requirement for CD4⁺ T cell help in the activation of naive CD8⁺ T cells (43, 44). Thus, the ability of FGK45 administration to promote the acquisition of effector T cell function among adoptively transferred TCR-I T cells following transfer into 501 mice was determined.

TCR-I cells were transferred into 501 mice treated with FGK45 or an isotype-matched control Ab. After 7 days, TCR-I cells isolated from cervical LNs and spleens were quantitated by staining with Db/I Tet and epitope I peptide-induced IFN- production. Line 501 mice treated with FGK45 accumulated 2- and 3-fold more TCR-I cells in cervical LNs and spleens, respectively, compared with mice that received control Ab (Fig. 5A). Importantly, treatment with FGK45 resulted in an increase in the fraction of TCR-I T cells that produced IFN- in response to epitope I peptide. The ratio of IFN--producing cells to Db/epitope I tetramer-positive cells in the LNs was 0.6 in FGK45-treated mice compared with 0.18 in control Ab-treated mice. Thus, treatment with FGK45 led to enhanced accumulation of TCR-I T cells in 501 mice that displayed increased effector function.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Treatment of 501 mice with anti-CD40 Ab enhances proliferation and induces effector function in TCR-I T cells. A, Five million clonotypic TCR-I cells were transferred into 6-mo-old 501 mice that also received 100 µg of purified FGK45 or rat IgG on the day before and the day after cell transfer. Seven days after transfer, lymphocytes were isolated from the spleen and cervical LNs and CD8+ T cells were stained with Db/epitope I tetramer (Db/I Tet) or stained for the production of IFN- following 6 h of in vitro stimulation with epitope I peptide. The number of epitope I-specific CD8+ T cells was quantitated by FACS analysis. The percentage of CD8+ T cells specific for epitope I is indicated in the upper right quadrant. Values were calculated by subtracting the percentage of CD8+ T cells that stained positive using a control tetramer or after stimulation with a control peptide, respectively. The samples shown are representative of three mice per group. B, Lymphocytes derived from line 416 mice were labeled with 5 µM CFSE, as described in Materials and Methods, before transfer of 5 x 106 clonotypic TCR-I cells into 6-mo-old 501 mice that were treated with FGK45 or control Ab. Groups of C57BL/6 mice also were given 5 x 106 CFSE-labeled TCR-I cells and either remained untreated or were immunized 1 day later with rVV-I. Eight days after transfer, CFSE staining was assessed among CD8+ Db/epitope I tetramer+ cells isolated from both spleen and cervical LNs. The percentage of cells that fall under each marker is indicated. C, Six-month-old 501 mice were injected with 100 µg FGK45 on days -1 and +1 or days +7 and +8 after transfer of 5 x 106 clonotypic TCR-I cells. The number of TCR-I cells re-isolated from cervical LNs and spleens of recipient mice were then analyzed at days 14 and 21 by staining with Db/epitope I tetramer (Db/I Tet) or peptide I-induced IFN- production. The percentage of CD8+ T cells specific for epitope I is indicated in the upper right quadrant. The values shown were corrected by subtracting the percentage of CD8+ T cells that stained positively with control tetramer or which produced IFN- following incubation with control peptide, respectively.

Although these results indicated that FGK45 treatment could promote the acquisition of T cell effector function if administered before the transfer of TCR-I T cells into 501 mice, a more realistic scenario was whether FGK45 treatment could promote the accumulation of effector T cells that had been previously exposed to the tolerogenic environment of 501 mice. Thus, the effect of administering FGK45 Ab 7 days posttransfer of TCR-I T cells was determined. This corresponds to a time point when TCR-I T cells had expanded in 501 mice, but failed to develop effector function (Fig. 3A). This approach resulted in the accumulation of similar numbers of TCR-I T cells in the draining LNs by day 14 postadoptive transfer whether or not FGK45 was administered before transfer of TCR-I T cells or at 7 days posttransfer (Fig. 5C, tetramer analysis, compare day -1, +1 to day 7, 8). By comparison, significantly more TCR-I T cells accumulated in the spleens of 501 mice if FGK45 was administered before cell transfer (62.4 vs 26.4% of CD8⁺ T cells). This difference was less apparent by day 21 posttransfer. The ratio of IFN--producing cells to Db/I⁺ CD8⁺ T cells also was reduced at day 14 in mice that received delayed treatment with FGK45 (LN, 0.31; spleen, 0.51) compared with mice pretreated with FGK45 (LN, 0.54; spleen, 0.96). This discrepancy was not apparent at day 21, perhaps reflective of the stable memory T cell pool. These results indicate that FGK45 treatment remained effective in promoting the acquisition of T cell function if administered after exposure of naive TCR-I T cells to the toleragenic environment of 501 mice and established a stable memory T cell population capable of producing IFN- in response to Ag.

Some FGK45-treated 501 mice that received TCR-I T cells at 6 mo of age were monitored for the long-term maintenance of epitope I-specific T cells. Three of five mice retained detectable levels of epitope I-specific CD8⁺ T cells in the peripheral blood up to 1 year postadoptive transfer, ranging from 3–18% of CD8⁺ T cells (data not shown). Analysis of lymphocytes derived from spleen and cervical LNs with MHC tetramers revealed similar frequencies of epitope I-specific CD8⁺ T cells in these mice. In addition, a proportion of these epitope I-specific CD8⁺ T cells were capable of producing IFN- in response to epitope I peptide stimulation in vitro (data not shown). Thus, FGK45 treatment is associated with the long-term survival of TCR-I T cells in 501 mice.

TCR-I T cells from anti-CD40-treated 501 mice have in vivo effector function

A classic function of a CD8⁺ T cell is to lyse a target cell expressing its cognate Ag. To test the ability of TCR-I cells that encountered endogenous T Ag in 501 mice to destroy target cells in vivo, the fate of epitope I peptide-pulsed spleen cells was monitored following injection into 501 mice previously given TCR-I T cells. Target cells were obtained from congenic B6/SJL mice, which express the CD45.1 molecule that allowed the donor cells to be distinguished from the recipient CD45.2-expressing lymphocytes by staining with anti-CD45.1 mAb. B6/SJL spleen cells were pulsed with either T Ag epitope I peptide or influenza NP 366–374 peptide. Epitope I-pulsed cells were subsequently labeled with a high concentration of CFSE while NP366–374 pulsed cells were labeled with a 10-fold lower concentration of CFSE. These cells were subsequently mixed in equal proportions and 5 x 10⁶ total cells were injected into 501 or C57BL/6 mice that had previously received TCR-I cells 14 days earlier with or without FGK45 treatment. After 6 h, spleens were harvested and analyzed for the presence of CD45.1⁺, CFSE⁺ cells. As shown in Fig. 6, epitope I-pulsed (CFSE^high) cells were undetectable in the spleens of 501 mice that were treated with FGK45, whereas the low CFSE-labeled cells pulsed with the control peptide remained. In contrast, equal proportions of high and low CFSE-labeled CD45.1 lymphocytes were detected in the spleens of 501 mice given control Ab, indicating the absence of significant epitope I-specific lytic activity. Control C57BL/6 recipients maintained both high and low CFSE-labeled populations of target cells, demonstrating that FGK45 treatment in the absence of endogenous T Ag does not lead to activation of the TCR-I T cells. In contrast, epitope I-pulsed target cells were eliminated in TCR-I T cell recipient C57BL/6 mice that were previously immunized against epitope I. These results demonstrate that FGK45 treatment of 501 mice leads to the activation of adoptively transferred TCR-I T cells that develop potent in vivo epitope-specific lytic activity.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. TCR-I T cells transferred into 501 mice treated with anti-CD40 Ab are cytotoxic in vivo. TCR-I T cells were transferred into 6-mo-old 501 (A) or C57BL/6 (B) mice that received 100 µg FGK45 or an isotype-matched control Ab the day before and the day after cell transfer. An additional group of C57BL/6 mice received TCR-I T cells and subsequent immunization with B6/K-TagI cells that express a T Ag variant in which epitopes II/III, IV, and V are inactivated. After 14 days, mice were injected i.v. with a mixture of 2.5 x 106 T Ag epitope I peptide-pulsed B6/SJL (CD45.1+) spleen cells labeled with 5 µM CFSE and 2.5 x 106 Flu NP 366–374 peptide-pulsed B6/SJL spleen cells labeled with 0.5 µM CFSE. After 6 h, CD45.1+ cells derived from recipient spleens were examined by FACS analysis to quantitate the number of CFSEhigh and CFSElow cells that were recovered.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several approaches have been proposed to block or reverse the onset of peripheral T cell tolerance for the purpose of salvaging potentially therapeutic T cells within the host T cell repertoire. Because the role of the APC is central for complete activation of naive CD8⁺ T cells, triggering APC function via ligation of the CD40 receptor has received recent attention (67). This approach was shown to block the onset of peripheral T cell tolerance in vivo if agonistic anti-CD40 Ab was administered along with specific immunization (46, 47, 48, 68). The ability of anti-CD40 ligation to lead to the priming of CD8⁺ T cells against endogenous self-Ags or preexisting tumors, however, has yielded a different set of results. Kedl et al. (50) demonstrated that the administration of anti-CD40 Ab into tumor-bearing mice in the absence of specific immunization led to the enhanced deletion of tumor-specific CD8⁺ T cells. More recently, Hernandez et al. (69) found that administration of anti-CD40 enhanced the proliferation of TCR transgenic CD8⁺ T cells specific for an influenza virus hemagluttinin (HA) epitope in HA transgenic mice, but failed to result in the induction of effector function. In contrast to these studies, we demonstrate that administration of FGK45 into 501 mice resulted in increased proliferation of TCR-I T cells within the secondary lymphoid organs as well as the acquisition of effector functions, including the ability to destroy T Ag epitope I-expressing target cells in vivo. Although the basis for this difference is not readily apparent, some possible explanations may include differences in the antigenic systems utilized, different levels of endogenous Ag expressed, or differences in the route of delivery or amount of anti-CD40 Ab utilized. Hernandez et al. (69) demonstrated that CD8⁺ T cell effector function could be induced in HA-specific CD8⁺ T cells if activated HA-specific CD4⁺ T cells were coinjected or if IL-12 was coadministered with the anti-CD40 Ab. These results suggested that additional signals, such as cytokines, were required for the full activation and subsequent acquisition of effector functions by naive HA-specific TCR transgenic CD8⁺ T cells in HA transgenic mice. One possible explanation for the success of CD40 ligation alone to lead to the acquisition of effector function among naive TCR-I T cells is that the CD4⁺ T cell compartment might not be tolerant in 501 mice, in contrast to the HA transgenic mice, such that additional signals could be provided by the endogenous CD4⁺ T cells in 501 mice to either the APCs or the TCR-I T cells. Preliminary results in 501 mice, however, indicate that the presence of CD4⁺ T cells is not required for anti-CD40 administration to promote the acquisition of effector function by TCR-I T cells (data not shown). An alternative explanation is that the TCR-I T cells might not require additional signals other than those delivered by CD40-induced APCs. Future studies will assess the phenotype of CD40-induced APCs from 501 mice to determine whether these APCs alone are fully capable of activating naive TCR-I T cells.

Differences in the level of Ag expression or the antigenic properties of the Ag systems investigated might also explain the observed differences. Previous investigations assessing the ability of H-2-K^k-restricted, T Ag epitope specific TCR transgenic CD8⁺ T cells to respond to endogenous T Ag expressed from the insulin promoter demonstrated that transgenic expression of the full-length T Ag (70), but not a nontransforming truncated protein (26), led to the induction of autoimmunity. These results suggested that the oncogenic process initiated by full-length T Ag might increase the priming of autoreactive T cells, while expression of a nontransforming mutant T Ag resulted in immunologic ignorance (26). Thus, the full-length T Ag might be capable of providing additional signals to the APCs or other immune effector cells, such that CD40 ligation alone results in full activation of the naive CD8⁺ T cells in 501 mice.

A few studies have indicated that the administration of anti-CD40 Ab can lead to a more rapid onset of tolerance (50, 71). Our analysis indicates that TCR-I T cells exposed to the endogenous T Ag for one week remained responsive to the activating effects of anti-CD40 administration. Whether these T cells will remain responsive for longer periods of time is unknown, but our data indicate that at least some of the T cells can be rescued by anti-CD40 administration. One potential difference between our study and those that showed anti-CD40-induced deletion of responsive T cells is that the mice in previous studies were given multiple injections of anti-CD40 Ab over the course of the experiment vs two doses of FGK45 at 1-day intervals administered in this study. Mauri et al. (71) assessed the effects of anti-CD40 ligation on the development of collagen-induced arthritis following repeated injections of anti-CD40 into collagen type II-treated animals. These authors found that repeated anti-CD40 administration decreased the onset of disease severity and skewed the T cell response from Th type 1 to type 2. They suggested that anti-CD40 treatment might skew the dominant APC from DCs to B cells, which favor a Th type 2 response rather than a Th type I response. Thus, repeated injections of anti-CD40 might be detrimental for maintaining CD8⁺ T cell responses, suggesting that the timing of anti-CD40 administration needs to be carefully assessed.

One important aspect of the present study is the finding that administration of anti-CD40 led to the establishment of a stable memory population of functional TCR-I T cells on a systemic level. High levels of TCR-I T cells were detected in 501 mice by day 21 postadoptive transfer regardless of whether the mice were treated with anti-CD40 at the time of TCR-I T cell transfer or 1 wk later. In addition, a significant proportion of these T cells maintained the ability to produce IFN- upon stimulation in vitro. We also found that these T cells could be detected in the peripheral blood of anti-CD40 treated 501 mice up to 1 year following adoptive transfer, ranging from 3 to 18% of the total CD8⁺ T cells. This finding is particularly intriguing in the light of recent studies that demonstrated that CD4⁺ effector T cells and CD8⁺ memory T cells remained susceptible to the effects of peripheral tolerance to self-Ag in HA transgenic mice (72, 73). We have yet to determine whether the TCR-I long-term memory T cells in 501 mice can effectively control tumor progression in 501 mice or maintain the ability to destroy epitope I-expressing target cells in vivo. These memory T cells, however, retained the ability to produce IFN- following in vitro stimulation (data not shown). Thus, effector T cells generated in the presence of anti-CD40 treatment establish a stable memory T cell pool that might later be tapped for immunotherapy of developing tumors.

In conclusion, the results presented in this study provide encouraging support for the use of anti-CD40 treatment to generate significant CD8⁺ T cell responses to tumor-associated self-Ags. Clearly the conditions for anti-CD40 treatment must be carefully evaluated for each particular Ag system to provide maximal stimulatory responses and prevent accelerated deletion of responding T cells. Key to this approach is the presence of a population of T cells capable of responding to the target Ag, which might consist of the residual endogenous T cell response, or be derived from adoptive transfer. Thus, in cases where T cell precursors are limiting, the use of specific immunization or ex vivo expansion to generate increased numbers of effector T cells in combination with in vivo anti-CD40 treatment might be desirable.

	Acknowledgments

 
We thank Melanie Epler, Andrew Gaydos, and Rebecca Pound for excellent technical assistance.

	Footnotes

 
¹ This work was supported by research Grants CA-25000 (to S.S.T.) and CA-24694 (to M.J.T.) from the National Cancer Institute, National Institutes of Health, and the Franklin Martin, MD, FACS Memorial Faculty Research Award from The American College of Surgeons (to K.S.-O.), and a grant from the Pennsylvania State University Cancer Center (to K.S.-O.).

² Current address: Department of Natural Sciences, Messiah College, Box 3030, Grantham, PA 17027.

³ Address correspondence to Dr. Satvir S. Tevethia, Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033. E-mail address: sst1{at}psu.edu

⁴ Abbreviations used in this paper: T Ag, large tumor Ag; DC, dendritic cell; LN, lymph node; NP, nucleoprotein; Flu, influenza virus; HA, hemagglutinin; rVV, recombinant vaccinia virus; TCR-I, epitope I-specific TCR transgenic.

Received for publication April 4, 2003. Accepted for publication May 6, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Boon, T., P. van der Bruggen. 1996. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183:725.[Free Full Text]
2. Wang, R. F., S. A. Rosenberg. 1996. Human tumor antigens recognized by T lymphocytes: implications for cancer therapy. J. Leukocyte Biol. 60:296.[Abstract]
3. Theobald, M., J. Biggs, J. Hernandez, J. Lustgarten, C. Labadie, L. A. Sherman. 1997. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J. Exp. Med. 185:833.[Abstract/Free Full Text]
4. Schell, T. D., L. M. Mylin, I. Georgoff, A. K. Teresky, A. J. Levine, S. S. Tevethia. 1999. Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice. J. Virol. 73:5981.[Abstract/Free Full Text]
5. Colella, T. A., T. N. Bullock, L. B. Russell, D. W. Mullins, W. W. Overwijk, C. J. Luckey, R. A. Pierce, N. P. Restifo, V. H. Engelhard. 2000. Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy. J. Exp. Med. 191:1221.[Abstract/Free Full Text]
6. Zheng, X., J. X. Gao, H. Zhang, T. L. Geiger, Y. Liu, P. Zheng. 2002. Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors. J. Immunol. 169:4761.[Abstract/Free Full Text]
7. Stockinger, B.. 1999. T lymphocyte tolerance: from thymic deletion to peripheral control mechanisms. Adv. Immunol. 71:229.[Medline]
8. Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19:47.[Medline]
9. Brinster, R. L., H. Y. Chen, A. Messing, T. van Dyke, A. J. Levine, R. D. Palmiter. 1984. Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors. Cell 37:367.[Medline]
10. Palmiter, R. D., H. Y. Chen, A. Messing, R. L. Brinster. 1985. SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumors in transgenic mice. Nature 316:457.[Medline]
11. Butel, J. S., J. A. Lednicky. 1999. Cell and molecular biology of simian virus 40: implications for human infections and disease. J. Natl. Cancer Inst. 91:119.
12. Tevethia, S. S., T. D. Schell. 2001. The immune response to SV40, JCV and BKV. K. Khalili, and G. L. Stoner, eds. Human Polyomaviruses: Molecular and Clinical Perspectives 585. Wiley-Liss, New York.
13. Deckhut, A. M., J. D. Lippolis, S. S. Tevethia. 1992. Comparative analysis of core amino acid residues of H-2D^b-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen. J. Virol. 66:440.[Abstract/Free Full Text]
14. Mylin, L. M., R. H. Bonneau, J. D. Lippolis, S. S. Tevethia. 1995. Hierarchy among multiple H-2^b-restricted cytotoxic T lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69:6665.[Abstract]
15. Mylin, L. M., A. M. Deckhut, R. H. Bonneau, T. D. Kierstead, M. J. Tevethia, D. T. Simmons, S. S. Tevethia. 1995. Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2K^b-restricted determinant in simian virus 40 tumor antigen. Virology 208:159.[Medline]
16. Faas, S. J., S. Pan, C. A. Pinkert, R. L. Brinster, B. B. Knowles. 1987. Simian virus 40 (SV40)-transgenic mice that develop tumors are specifically tolerant to SV40 T antigen. J. Exp. Med. 165:417.[Abstract/Free Full Text]
17. Jolicoeur, C., D. Hanahan, K. M. Smith. 1994. T cell tolerance towards a transgenic -cell antigen and transcription of endogenous pancreatic genes in the thymus. Proc. Natl. Acad. Sci. USA 91:6707.[Abstract/Free Full Text]
18. Antonia, S. J., T. Geiger, J. Miller, R. A. Flavell. 1995. Mechanisms of immune tolerance induction through the thymic expression of a peripheral tissue-specific protein. Int. Immunol. 7:715.[Abstract/Free Full Text]
19. Schell, T. D., B. B. Knowles, S. S. Tevethia. 2000. Sequential loss of cytotoxic T lymphocyte responses to simian virus 40 large T antigen epitopes in T antigen transgenic mice developing osteosarcomas. Cancer Res. 60:3002.[Abstract/Free Full Text]
20. Knowles, B. B., J. McCarrick, N. Fox, D. Solter, I. Damjanov. 1990. Osteosarcomas in transgenic mice expressing an -amylase-SV40 T-antigen hybrid gene. Am. J. Pathol. 137:259.[Abstract]
21. Marton, I., S. E. Johnson, I. Damjanov, K. S. Currier, J. P. Sundberg, B. B. Knowles. 2000. Expression and immune recognition of SV40 Tag in transgenic mice that develop metastatic osteosarcomas. Transgenic Res. 9:115.[Medline]
22. Oehen, S. U., P. S. Ohashi, K. Burki, H. Hengartner, R. M. Zinkernagel, P. Aichele. 1994. Escape of thymocytes and mature T cells from clonal deletion due to limiting tolerogen expression levels. Cell. Immunol. 158:342.[Medline]
23. Ochsenbein, A. F., S. Sierro, B. Odermatt, M. Pericin, U. Karrer, J. Hermans, S. Hemmi, H. Hengartner, R. M. Zinkernagel. 2001. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 411:1058.[Medline]
24. Scheinecker, C., R. McHugh, E. M. Shevach, R. N. Germain. 2002. Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J. Exp. Med. 196:1079.[Abstract/Free Full Text]
25. Ohashi, P. S., S. Oehen, K. Buerki, H. Pircher, C. T. Ohashi, B. Odermatt, B. Malissen, R. M. Zinkernagel, H. Hengartner. 1991. Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 65:305.[Medline]
26. Soldevila, G., T. Geiger, R. A. Flavell. 1995. Breaking immunologic ignorance to an antigenic peptide of simian virus 40 large T antigen. J. Immunol. 155:5590.[Abstract]
27. Ye, X., J. McCarrick, L. Jewett, B. B. Knowles. 1994. Timely immunization subverts the development of peripheral nonresponsiveness and suppresses tumor development in simian virus 40 tumor antigen-transgenic mice. Proc. Natl. Acad. Sci. USA 91:3916.[Abstract/Free Full Text]
28. Kurts, C., H. Kosaka, F. R. Carbone, J. F. Miller, W. R. Heath. 1997. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8⁺ T cells. J. Exp. Med. 186:239.[Abstract/Free Full Text]
29. Staveley-O’Carroll, K., E. Sotomayor, J. Montgomery, I. Borrello, L. Hwang, S. Fein, D. Pardoll, H. Levitsky. 1998. Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc. Natl. Acad. Sci. USA 95:1178.[Abstract/Free Full Text]
30. Morgan, D. J., C. Kurts, H. T. Kreuwel, K. L. Holst, W. R. Heath, L. A. Sherman. 1999. Ontogeny of T cell tolerance to peripherally expressed antigens. Proc. Natl. Acad. Sci. USA 96:3854.[Abstract/Free Full Text]
31. Mellman, I., R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255.[Medline]
32. Adler, A. J., D. W. Marsh, G. S. Yochum, J. L. Guzzo, A. Nigam, W. G. Nelson, D. M. Pardoll. 1998. CD4⁺ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells. J. Exp. Med. 187:1555.[Abstract/Free Full Text]
33. den Haan, J. M. M., S. M. Lehar, M. J. Bevan. 2000. CD8⁺ but not CD8^- dendritic cells cross-prime cytotoxic T cells In vivo. J. Exp. Med. 192:1685.[Abstract/Free Full Text]
34. Belz, G. T., G. M. N. Behrens, C. M. Smith, J. F. A. P. Miller, C. Jones, K. Lejon, C. G. Fathman, S. N. Mueller, K. Shortman, F. R. Carbone, W. R. Heath. 2002. The CD8⁺ dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196:1099.[Abstract/Free Full Text]
35. Huang, F. P., N. Platt, M. Wykes, J. R. Major, T. J. Powell, C. D. Jenkins, G. G. MacPherson. 2000. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J. Exp. Med. 191:435.[Abstract/Free Full Text]
36. Kurts, C., R. M. Sutherland, G. Davey, M. Li, A. M. Lew, E. Blanas, F. R. Carbone, J. F. Miller, W. R. Heath. 1999. CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose. Proc. Natl. Acad. Sci. USA 96:12703.[Abstract/Free Full Text]
37. Morgan, D. J., H. T. Kreuwel, L. A. Sherman. 1999. Antigen concentration and precursor frequency determine the rate of CD8⁺ T cell tolerance to peripherally expressed antigens. J. Immunol. 163:723.[Abstract/Free Full Text]
38. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
39. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
40. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J. Exp. Med. 184:741.[Abstract/Free Full Text]
41. Schulz, O., A. D. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13:453.[Medline]
42. Moodycliffe, A. M., V. Shreedhar, S. E. Ullrich, J. Walterscheid, C. Bucana, M. L. Kripke, L. Flores-Romo. 1999. CD40-CD40 ligand interactions in vivo regulate migration of antigen-bearing dendritic cells from the skin to draining lymph nodes. J. Exp. Med. 191:2011.
43. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
44. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
45. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474.[Medline]
46. Diehl, L., A. T. den Boer, S. P. Schoenberger, E. I. van der Voort, T. N. Schumacher, C. J. Melief, R. Offringa, R. E. Toes. 1999. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy. Nat. Med. 5:774.[Medline]
47. Sotomayor, E. M., I. Borrello, E. Tubb, F. M. Rattis, H. Bien, Z. Lu, S. Fein, S. Schoenberger, H. I. Levitsky. 1999. Conversion of tumor-specific CD4⁺ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat. Med. 5:780.[Medline]
48. Bonifaz, L., D. Bonnyay, K. Mahnke, M. Rivera, M. C. Nussenzweig, R. M. Steinman. 2002. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8⁺ T cell tolerance. J. Exp. Med. 196:1627.[Abstract/Free Full Text]