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The Journal of Immunology, 2007, 179, 6686 -6695
Copyright © 2007 by The American Association of Immunologists, Inc.
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Anti-CD40 Conditioning Enhances the TCD8 Response to a Highly Tolerogenic Epitope and Subsequent Immunotherapy of Simian Virus 40 T Antigen-Induced Pancreatic Tumors1

Pavel Otahal2,*, Barbara B. Knowles{dagger}, Satvir S. Tevethia* and Todd D. Schell3,*

* Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033; and {dagger} The Jackson Laboratory, Bar Harbor, ME 04609


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rapid loss of adoptively transferred tumor-specific CD8+ T cells (TCD8) following Ag recognition in the periphery and their limited accumulation within the tumor stroma reduces the effectiveness of T cell-based immunotherapy. To better understand the role of TCD8 in the control of autochthonous tumors, we have used mice of the RIP1-Tag4 lineage that develop pancreatic β cell tumors due to expression of the SV40 large T Ag from the rat insulin promoter. We previously showed that the kinetics of functional TCD8 tolerance varies toward two distinct epitopes derived from T Ag. Epitope I (206SAINNYAQKL215)-specific TCD8 are rapidly deleted whereas TCD8 targeting epitope IV (404VVYDFLKC411) persist over the lifetime of tumor-bearing animals. In this report, we show that the conditioning of tumor-bearing RIP1-Tag4 mice with agonistic anti-CD40 Ab induces extensive expansion of naive epitope I-specific TCR transgenic (TCR-I) T cells in this tolerogenic environment and delays their loss from the host. In addition, functional TCR-I T cells intensively infiltrate pancreatic tumors, resulting in increased survival of RIP1-Tag4 mice. These results suggest that a similar approach could effectively enhance T cell-based immunotherapies to cancer when targeting other highly tolerogenic epitopes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
One obstacle to successful CD8+ T cell (TCD8)4 -mediated immunotherapy of autochthonous tumors is the inactivation or deletion of TCD8 with the potential to recognize tumor-associated epitopes presented by MHC class I molecules (1, 2). This can result from the elimination of self/tumor Ag-reactive T cells during T cell development and/or mechanisms that induce T cell tolerance in the periphery. The residual self-reactive T cell repertoire contains tumor Ag-specific precursors that can be activated by specific immunization or in vitro stimulation (3). This finding has been the premise for developing TCD8-based therapeutic approaches to cancer, such as adoptive immunotherapy that provides high numbers of effector T cells with the potential to destroy established tumors (4). However, these T cells may be susceptible to the effects of peripheral tolerance following the recognition of tumor-associated self-Ags in vivo (5, 6, 7). The extent of TCD8 peripheral tolerance toward tumor-derived epitopes varies, with some epitopes inducing more rapid tolerance than others (8, 9). To maximize the repertoire of potential tumor-reactive T cells, the effects of peripheral tolerance on T cells most susceptible to inactivation must be blocked. Thus, approaches that limit peripheral tolerance and promote T cell accumulation in the tumor microenvironment are needed to control the progression of advanced stage tumors.

Induction of TCD8 tolerance in the periphery can occur through a number of mechanisms including T cell recognition of bone marrow-derived APCs, which express self-Ags that can induce T cell deletion or anergy (10). Active suppression by regulatory T cells and the production of immunosuppressive cytokines within the tumor microenvironment can also limit tumor-specific immunity (11). One approach shown to prevent or delay the onset of peripheral TCD8 tolerance is the triggering of CD40 on host APCs (5, 12, 13, 14, 15, 16). CD40, a member of the TNF receptor family, is expressed on APCs such as B cells, macrophages, and dendritic cells as well activated T cells and endothelial cells (17). The triggering of CD40 on APCs through engagement of its ligand CD40L, which is found mainly on CD4+ T cells, or by using agonistic anti-CD40 Abs enhances APC-mediated priming of naive TCD8 against cross-presented Ag in the absence of CD4+ T cell help (18, 19, 20). This issue is particularly relevant when considering the priming of TCD8 toward established tumors. Systemic delivery of anti-CD40 Ab has been used to enhance TCD8 responses to tumors (21, 22, 23), but these studies did not address whether this approach could protect self/tumor-reactive TCD8 from peripheral tolerance to target autochthonous tumors.

Deletion of adoptively transferred TCD8 specific for the H-2Db-restricted SV40 T Ag epitope I (residues 206–215) was prevented in mice that develop T Ag-induced osteosarcomas when mice were preconditioned with an anti-CD40 agonist Ab (5). In that study, however, the effect of such treatment on tumor progression was not investigated due to the slow onset of tumor appearance. Recently, we demonstrated that epitope I-specific TCD8 also are highly susceptible to peripheral tolerance in a different SV40 T Ag transgenic lineage, line RIP1-Tag4 (RT4) mice (9), which express SV40 T Ag as a transgene from the rat insulin promoter and develop insulinomas (24, 25). T Ag expression begins around 5 wk of age in RT4 mice, leading to the development of islet hyperplasia by three months of age. Macroscopic insulinomas appear at 5 to 6 mo of age. The average life span of RT4 mice is 200 days and all mice eventually succumb to tumors due to hypoglycemia (25). Thus, RT4 mice provide a more suitable model to test the ability of anti-CD40 conditioning to modulate peripheral tolerance of epitope I-specific TCD8 and the resulting effect on tumor progression.

The immune response to T Ag in mice of the C57BL/6 background is characterized by a hierarchical TCD8 response to multiple epitopes (26). The H-2Kb-restricted response to epitope IV (T Ag 404–411) is the most dominant, followed by responses to the H-2Db-restricted T Ag epitopes I (T Ag 206–215; SAINNYAQKL) and II/III (T Ag 223–231) (27, 28). A fourth epitope, designated epitope V, is immunorecessive and does not induce TCD8 unless the three dominant epitopes are inactivated (29). In previous studies using T Ag transgenic mice, we demonstrated that epitope I-specific TCD8 are eliminated from the peripheral T cell repertoire more rapidly than TCD8 specific for epitope IV (8, 9). In addition, immunization toward epitope IV can prevent the development of insulinomas in RT4 mice, although this approach fails to mediate the regression of established tumors (9). In contrast, the ability to target epitope I is hampered by the rapid onset of peripheral tolerance for both the endogenous T cell population and adoptively transferred T cells (5, 9). The adoptive transfer of TCR transgenic TCD8 specific for epitope I (TCR-I) results in their initial proliferation in RT4 mice but not their accumulation in either the lymphoid tissues or the tumor (9). These cells disappear from RT4 mice within 10 days following adoptive transfer unless the mice also receive immunization at the time of adoptive transfer, indicating that naive TCD8 specific for epitope I are readily triggered against the endogenous tumor Ag in RT4 mice but are rapidly eliminated from the host.

The goal of the current study was to determine the extent to which TCR-I T cell survival could be enhanced in RT4 mice and whether these cells accumulate in the tumor, resulting in control of tumor progression. We find that rapid deletion of TCR-I T cells in RT4 mice can be delayed by the coadministration of agonistic anti-CD40 Ab. These TCR-I T cells proliferate extensively in RT4 mice after treatment with anti-CD40, acquire effector function, and intensively infiltrate pancreatic tumors, correlating with an increased lifespan for RT4 mice. These results demonstrate that therapeutic effects can be achieved even with TCD8 that are highly susceptible to peripheral tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6J (H2b), mice were purchased from The Jackson Laboratory and maintained at the animal facility of the Milton S. Hershey Medical Center (Hershey, PA) and used between 8 and 16 wk of age. TCR-I mice on the C57BL/6J background were described previously (5) (B6.Cg-Tg(TcraTcrb)416Tev/J; available from The Jackson Laboratory). Progeny were screened by staining PBL with anti-Vβ7 Ab (BD Pharmingen). Line RT4 mice are on the C57BL/6J background and express the SV40 early region under the control of the rat insulin promoter (24). B6.SJL mice were purchased from Taconic Farms. Ubiquitin C-GFP mice expressing GFP from the ubiquitin promoter (30) were purchased from The Jackson Laboratory. In some experiments, the F1 generation of TCR-I males bred with B6.SJL females and the F1 generation of TCR-I females bred with ubiquitin C-GFP males were used. All animal studies were performed in accordance with guidelines established by the Pennsylvania State University College of Medicine Animal Care and Use Committee (Hershey, PA) under an approved protocol.

Cell lines, medium, and peptides

The B6/WT-19 cell line was derived by the transformation of primary C57BL/6 mouse embryo fibroblasts with the wild-type SV40 strain VA45-54 and expresses full length T Ag (31). Cell lines were maintained in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin, 2 mM L-glutamine, 10 mM HEPES, 0.075% (w/v) NaHCO3, and 5–10% FBS. 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). Peptides were solubilized in DMSO and diluted to the appropriate concentration with RPMI 1640 medium. Peptides used in these experiments correspond to the SV40 T Ag epitope I (SAINNYAQKL; peptide I) and influenza virus (Flu) nucleoprotein (NP) 366–374 (ASNENMETM; peptide Flu).

Flow cytometric analysis of lymphocyte populations

Ex vivo staining of lymphocytes with MHC tetramers and primary conjugated Abs was performed on a splenic single-cell suspension as described previously (28). Cells were fixed after staining with 2% paraformaldehyde in PBS, and analyzed using a FACScan flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star). Production and characterization of the H-2Db/T Ag epitope I (Db/I), H-2Db/T Ag epitope V (Db/V), H-2Kb/T Ag epitope IV (Kb/IV), and H-2Db/Flu NP epitope 366–374 (Db/Flu) tetramers was previously described (28). The following Abs were purchased from BD Pharmingen: PE-labeled and PE-Cy5-labeled rat anti-CD8a (clone 53-6-7), FITC-labeled rat anti-mouse CD44 (clone IM7), PE-labeled mouse anti-mouse CD45.1 (clone A20), FITC-labeled hamster anti-mouse CD69 (clone H1.2F3), and FITC-labeled rat anti-mouse IFN-{gamma} (clone XMG1.2). The percentage of CD8+ T cells that stained positive with a T Ag-specific tetramer was determined by subtracting the percentage of cells that stained positive with the control Db/Flu tetramer within the same population.

Adoptive transfers, immunizations, Ab treatment, and blood glucose measurement

Mice received the adoptive transfer of sex-matched RBC-depleted lymphocytes derived from spleens and lymph nodes of TCR-I transgenic mice. Recipients were injected i.v. in the tail vein with a cell suspension containing 1–10 x 106 clonotypic TCR-I T cells, as determined by tetramer staining, in 0.2 ml HBSS. One day before and one day after adoptive transfer the mice received 100 µg (i.p. in 0.2 ml of PBS) of the purified anti-CD40 Ab FGK45 (32) or control rat IgG (Sigma-Aldrich). Immunization of mice with T Ag-expressing cell lines was conducted by the injection of 3 x 107 cells (i.p. in 0.5 ml of HBSS). Glucose levels from tail vein-derived peripheral blood were measured by using an Elite glucometer (Bayer).

In vivo cytotoxicity and proliferation assay

Sex-matched C57BL/6J splenocytes were incubated in the presence of the indicated peptides (1 µM) in RPMI 1640 with 10% FBS at 37°C for 90 min and washed three times with cell medium. Peptide-coated targets were then labeled with different concentrations (5 µM CFSEhigh and 0.5 µM CFSElow) of 5- and 6-CFSE (Molecular Probes) for 10 min at 37°C in PBS with 0.1% BSA, washed two times, mixed together at a 1:1 ratio, and then 4 x 106 cells were injected into the tail vein (0.2 ml in HBSS). Elimination of CFSE-labeled target cells was assessed after 16 h. The in vivo proliferation assay was performed by i.v. injection of RBC-depleted lymphocytes from TCR-I mice that were first labeled with 5 µM-CFSE for 10 min at 37°C. After washing cells three times with PBS, cells were resuspended in HBSS and injected i.v. at a dose of 5 x 106 clonotypic TCR-I T cells per mouse. The proliferation of injected cells was determined 3 days later by staining the cell suspension prepared from spleen and cervical, and pancreatic lymph nodes with anti-CD8 Ab and Db/I tetramer. The extent of proliferation was determined by measuring the mean fluorescence intensity of CFSE dye in CD8+Db/I tetramer+ cells.

In situ tetramer staining and tumor histology

In situ staining of tissues with MHC tetramers was performed using a modification of a previously described method (33). Native tumor samples were cut into 3- to 4-mm pieces and stained overnight at 4°C in PBS and 2% FBS containing PE-labeled tetramers (1/10). After washing with PBS for 2 h at 4°C, the tissues were fixed with 4% paraformaldehyde for 1 h at room temperature and snap frozen. Cryostat-cut, 10-µm-thick sections were exposed for 1 h at room temperature to rat anti-CD8 Ab (1 µg/ml) and rabbit anti-PE serum (Biomeda) diluted 1/1000 in PBS with 2% normal goat serum. After washing with PBS, slides were then incubated with Alexa Fluor 555 goat anti-rabbit and Alexa Fluor 647 goat anti-rat Abs (Molecular Probes) diluted 1/500 in PBS and 2% goat serum containing 4',6'-diamidino-2-phenylindole (DAPI; 1 µg/ml) for 1 h at room temperature. To demonstrate the specificity of the staining, we analyzed sections prepared from spleens of TCR-I transgenic mice and B6 mice (see Fig. 3C). The Db/I tetramer bound specifically to the TCD8 in the TCR-I mice, whereas no tetramer staining was detected in the control B6 mice.


Figure 3
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  		FIGURE 3. Visualization of tumor-infiltrating TCR-I T cells. Groups of three RT4 mice aged 5 to 6 mo were injected with 1 x 106 TCR-I T cells expressing GFP (TCR-I.GFP) and treated with FGK45 the day before and after T cell injection. After 10 days, tumors were harvested and sections were stained and analyzed by confocal microscopy as described in Materials and Methods. A, TCR-I.GFP CD8+ T cells are detected within the tumor stroma and closely associate with insulin-expressing cells (shown in red). The areas outlined with white boxes are depicted at higher magnification in the right panels, which show DAPI staining of nuclei along with anti-CD8 staining (blue) of the TCR-I.GFP cells. B, Costaining of tumor cells for insulin (red) and T-Ag (blue). C, The specificity of the in situ tetramer staining is demonstrated on sections prepared from spleens of TCR-I transgenic mice (top row) and B6 mice (bottom row). The Db/I tetramer (green) bound specifically to the CD8+ cells (red) in the TCR-I mice, which is shown by the colocalization of both colors, whereas no tetramer staining or colocalization was detected in the control B6 mice. D, The presence of epitope I-specific TCR on GFP+ T cells is demonstrated by in situ tetramer staining with Db/I tetramer (red, top and middle row), whereas no staining was observed with control Db/Flu tetramer (bottom row). Scale bars, 40 µm (A–C) and 10 µm (D).

 
Fluorescent staining to detect the presence of GFP+ TCR-I T cells in the tumors was performed on 10-µm-thick frozen sections prepared from paraformaldehyde-fixed tumors. Sections were then incubated with rat anti-CD8 Ab (1 µg/ml) and guinea pig anti-insulin Ab (DakoCytomation) in PBS with 2% goat serum for 1 h at room temperature. After washing with PBS, the primary Abs were detected with Alexa Fluor 647 goat anti-rat Ab and Rhodamine Red-X donkey anti-guinea pig Ab (Jackson ImmunoResearch Laboratories) diluted in PBS and 2% goat serum containing DAPI (1 µg/ml). For simultaneous detection of insulin and T Ag expression, paraformaldehyde-fixed tumors were embedded in OCT medium and snap frozen in liquid nitrogen. Sections cut in a cryostat were then treated with 2 M HCl for 1 h at 37°C followed by incubation with mouse Ig-blocking solution (Vector Laboratories) for 1 h at room temperature. Sections were then stained with guinea pig anti-insulin and mouse anti-T Ag 901 mAb (34) for 45 min at room temperature in PBS, 2% normal goat serum, and 0.1% Nonidet P-40. After washing, the primary Abs were visualized with Rhodamine Red-X donkey anti-guinea pig Ab and Cy5-labeled goat anti-mouse Ab. After washing with PBS, all stained sections were then fixed with 4% paraformaldehyde for 10 min, mounted in Aqua-Poly/Mount (Polysciences), and examined with a Leica AOBS confocal microscope using a x40 immersion objective (Leica Microsystems). Images obtained by sequential scanning were processed and overlaid in Adobe Photoshop (Adobe Systems).

Statistical analysis

Kaplan-Meier survival plots were constructed with Prism software (GraphPad Software) and statistical significance was determined by the log rank posttest.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Conditioning RT4 mice with activating anti-CD40 FGK45 Ab increases accumulation of TCR-I T cells following recognition of endogenous T Ag

In a previous study (9), we found that TCR-I T cells are initially activated and induced to proliferate following transfer into RT4 mice but are rapidly deleted. In the present study, we addressed whether administering the anti-CD40 agonistic Ab, FGK45 (19), could rescue TCR-I T cells from deletion. This approach has been shown to overcome the development of tolerance in several experimental models (5, 6, 13) and is indicated as being mediated via activation of host APCs (35). Thus, we adoptively transferred TCR-I T cells (CD45.1 positive) into 5-wk-, 3-mo-, and 6-mo-old RT4 mice and control B6 mice. One day before and one day after adoptive transfer, the mice were treated with FGK45 or control rat IgG. Ten days after T cell transfer, the splenic T cells were stained with anti-CD8, anti-CD45.1, and Db/I tetramer to determine the total number of TCR-I T cells per spleen. The results in Fig. 1A show that treatment of RT4 mice with FGK45 resulted in the extensive accumulation of transferred TCR-I T cells compared to treatment with the control Ab. Accumulation reached similar levels in all groups of RT4 mice, suggesting that it was independent of the age of the RT4 mice. We observed 4.2-, 7.2- and 5.9–fold increases in TCR-I T cell accumulation when transferred into 5-wk-, 3-mo-, and 6-mo old RT4 mice, respectively, compared with the levels achieved in Ag-free B6 mice. Control IgG-treated RT4 mice at all three ages had reduced levels of TCR-I T cells compared with B6 mice by 10 days after adoptive transfer, indicative of T cell deletion in the absence of anti-CD40 conditioning.


Figure 1
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  		FIGURE 1. Application of agonistic anti-CD40 Ab FGK45 promotes strong expansion of adoptively transferred TCR-I T cells in RT4 mice. A, Groups of two to three RT4 mice aged 6 mo, 3 mo, and 5 wk and B6 control mice were treated with 100 µg of either FGK45 Ab or control Rat IgG. The next day, the same mice received 1 x 107 TCR-I T cells followed 1 day later by a second dose of the indicated Ab. The total number of TCR-I T cells was enumerated by tetramer staining of splenic lymphocytes 10 days after adoptive transfer. SD values are indicated. The numbers in parentheses indicate the percentage of CD8+ cells specific for epitope I. B, Three-month-old RT4 mice that received 3 x 106 CFSE-labeled TCR-I T cells were injected with FGK45 or control rat IgG 1 day before and 1 day after adoptive transfer. Three days after adoptive transfer the proliferation of TCR-I T cells was assessed in the spleens by tetramer staining. Values in each dot plot indicate the percentage of CD8+ cells specific for epitope I. CFSE intensity is shown as histograms for cells within the gated region of the dot plots. Values in the histograms indicate the mean fluorescence intensity of CFSE. Data are representative of two mice per group.

 
The enhanced TCR-I T cell accumulation observed following the conditioning of RT4 mice with FGK45 could be explained by the more rapid proliferation of TCR-I T cells. To address this possibility, CFSE-labeled TCR-I T cells were transferred into 3-mo-old RT4 mice with or without administration of FGK45, and the extent of proliferation was examined 3 days after adoptive transfer. The data in Fig. 1B show that the administration of FGK45 results in a robust accumulation of TCR-I T cells in just 3 days, representing 30% of CD8+ T cells, and that all of these cells had undergone multiple divisions (mean fluorescence intensity of 348). In contrast, there was no increase in TCR-I T cells in mice that received the control IgG at this time point, and the detectable cells had undergone fewer divisions within the same time period (mean fluorescence intensity of 452). These data indicate that all of the TCR-I T cells were activated in the absence of FGK45, but the T cells that had undergone more than seven divisions, resulting in loss of CFSE fluorescence, failed to accumulate in mice given the control Ab. This result could be explained by either slower proliferation or the abortive death of TCR-I T cells after four to five divisions in the absence of FGK45 conditioning. However, no difference in the percentage of apoptotic TCR-I T cells was detected among cells recovered from FGK45- or rat IgG-treated RT4 mice 3 days after adoptive transfer (data not shown). Taken together, these data suggest that FGK45 conditioning results in accelerated proliferation of TCR-I T cells following recognition of the endogenous T Ag, with ~10-fold expansion in 3 days.

TCR-I T cells infiltrate tumors in RT4 mice after administration of FGK45

Because conditioning RT4 mice with FGK45 induces an extensive accumulation of TCR-I T cells in the spleen, we determined whether these T cells migrated into tumors in RT4 mice. TCR-I T cells were transferred into tumor-bearing, 6-mo-old RT4 mice that were also given FGK45 or control rat IgG. Ten days later, pancreatic β cell tumors of ~5–8 mm in diameter were excised, cut into pieces and enzymatically digested to yield cell suspensions that could be analyzed for the presence of TCR-I cells by tetramer staining using flow cytometry (Fig. 2A). RT4 mice treated with control rat IgG+ TCR-I T cells or with FGK45 without adoptive transfer of TCR-I T cells contained few total TCD8 in their tumors (Fig. 2A, left panels). In comparison, RT4 mice that received TCR-I T cells and FGK45 contained a significant number of tumor-infiltrating TCD8, with ~20% of total cells representing epitope I-specific T cells.


Figure 2
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  		FIGURE 2. Conditioning with FGK45 induces tumor infiltration and induction of TCR-I T cell function in RT4 mice. A, Six-month-old RT4 mice received 1 x 106 or 1 x 104 SJL/TCR-I T cells (CD45.1+) plus 100 µg of FGK45 or control rat IgG the day before and the day after the transfer. A third group of mice received FGK45 alone without the adoptive transfer of TCR-I T cells. Ten days after adoptive transfer, mice were sacrificed, pancreatic tumors were enzymatically dissociated, and the resulting cell suspensions were stained with the indicated MHC tetramers and anti-CD8. The numbers shown in the dot plots indicate the percentage of total cells in each quadrant. Data shown are from a mouse that received 1 x 106 donor TCR-I cells and are representative of five mice. Similar results were obtained from mice that received either dose of TCR-I T cells. B, RT4 mice treated similarly as in A were subjected to an in vivo killing assay. Ten days after the adoptive transfer a mixture of peptide-pulsed, CFSE-labeled target cells were injected into treated RT4 mice and untreated B6 mice. The population of CFSEhigh targets corresponds to cells incubated with epitope I peptide, whereas CFSElow targets correspond to cells incubated with control peptide NP366–374. C and D, Four- to five-month-old RT4 mice received donor CD45.1+ TCR-I T cells and FGK45 as in A. Ten days after adoptive transfer, suspensions prepared from spleens and tumors of representative mice were stained with anti-CD8, MHC tetramer, and anti-CD45.1 (C) or subjected to intracellular cytokine staining after a 6-h stimulation with the indicated peptide (D). Data from the spleens of two individual mice are shown. Cells derived from the tumors of three mice were pooled before analysis. The values in each dot plot indicate the percentage of CD8+ cells that stained with either MHC tetramer or anti-IFN-{gamma}. The histograms in C show CD45.1 expression on the CD8+ Db/I tetramer+ (Tet+; top region, heavy line) and Db/I tetramer– (Tet–; bottom region, normal line) cells. This experiment was repeated with similar results.

 
To assess whether TCR-I T cells that accumulated in response to conditioning with FGK45 had gained effector functions in tumor-bearing RT4 mice, an in vivo killing assay was used. We found that peptide I-pulsed targets were efficiently eliminated in FGK45-treated RT4 mice but not in RT4 mice receiving control Rat IgG (Fig. 2B). To specifically test the function of the tumor-infiltrating TCR-I cells, IFN-{gamma} production was assessed by intracellular cytokine staining. To differentiate the donor lymphocytes from the host TCD8, we used CD45.1+ TCR-I T cells. At 10 days after adoptive transfer, epitope I-specific TCD8 were detected by both MHC tetramer staining (Fig. 2C) and intracellular cytokine staining (Fig. 2D) in parallel samples. The data show that the Db/I tetramer+ cells detected in the spleens and pancreatic tumors of RT4 mice were of donor origin, as shown by CD45.1 expression, whereas the Db/I tetramer negative fraction contained only host-derived cells (Fig. 2C, histograms). In addition, epitope I-specific IFN-{gamma} production was detected in both the spleens and tumors of these mice, demonstrating the presence of functional epitope I-specific T cells within the tumor. We found that the frequency of splenic TCD8 that produced IFN-{gamma} was approximately half that detected by Db/I tetramer staining but represented only a quarter of the Db/I tetramer+ cells detected in the tumor (Fig. 2D). Thus, while functional TCR-I TCD8 were detected in both the spleen and the tumor of RT4 mice, not all epitope I-specific TCD8 were able to produce IFN-{gamma} after their recovery at this time point.

Previous studies in a related line of T Ag transgenic mice that develop insulinomas revealed that activated T lymphocytes accumulate at the periphery of T Ag-expressing islets unless a secondary trigger is also provided, such as CpG or irradiation, resulting in T cell infiltration into the tumor stroma (36, 37). Recent clinical observations also indicate that CD8+ T cell infiltration of tumors correlates with a positive prognosis (38, 39). To determine whether TCR-I T cells accumulate within the tumor stroma after administration of FGK45, GFP+ TCR-I T cells were transferred into 6-mo-old RT4 mice that were also given FGK45. After 10 days, tumor sections were examined by confocal microscopy for the presence of GFP+ TCD8. Sections fixed with paraformaldehyde were costained for insulin and CD8 to localize TCD8 within the tumors (Fig. 3A). The results demonstrate that the GFP-expressing TCR-I T cells are localized within the insulin-producing stroma as well as outside of this area. Higher magnification revealed a close interaction between CD8+GFP+ cells and insulin-producing tumor cells (Fig. 3A, right panels). To demonstrate that tumor cells expressing insulin simultaneously express T Ag, we costained the sections with anti-insulin and anti-T Ag Ab after performing the epitope retrieval approach (Fig. 3B). The images show that insulin (shown in red) was detected in the cytoplasm whereas the T Ag (shown in blue) was detected in the nucleus. This result indicates that the insulin-positive cells within the tumor stroma also express T Ag.

To further confirm that the GFP+CD8+ cells detected within the tumors were epitope specific, we performed in situ tetramer staining. To first evaluate the specificity of the in situ tetramer technique, the spleens of TCR-I and B6 mice were stained with Db/I tetramer (Fig. 3C). The images show that the Db/I tetramer (shown in green) stained specifically the CD8+ cells (shown in red) in the TCR-I mice (top row) but not in the control B6 mice (bottom row). The same tumor samples as shown in Fig. 3A were then stained using an in situ tetramer detection technique. The images in Fig. 3D demonstrate that the Db/I tetramer, but not the control Db/Flu tetramer, colocalized with CD8 on the surface of the GFP+ cells. In conclusion, histological analysis of tumor sections demonstrates that TCR-I T cells activated by FGK45 conditioning infiltrate the tumors and closely associate with tumor cells to mediate the anti-tumor immune response.

Persistence of TCR-I T cells in RT4 mice after treatment with FGK45

We observed that conditioning with FGK45 induces vigorous expansion of adoptively transferred TCR-I T cells. To determine how long TCR-I T cells persist, FGK45-treated RT4 mice were given adoptive transfers with TCR-I T cells. Mice were assessed at 10 and 20 days after adoptive transfer for the presence of TCR-I T cells in the spleens and pancreatic tumors. The number of TCR-I T cells recovered was compared with B6 mice that were adoptively transferred with TCR-I T cells followed by immunization with B6/WT-19 T Ag transformed-fibroblasts expressing the wild-type T Ag. The data in Fig. 4A show that TCR-I T cells are detected at high levels in both the spleen (15.5% of TCD8) and the tumor (20.5% of TCD8) at day 10, comparable to the frequency induced in B6 mice following immunization (22% of TCD8). However, by 20 days after adoptive transfer the frequency of TCR-I T cells was reduced to 0.15% in the spleen and 2.6% in the tumor, whereas the number of TCR-I T cells detected in B6 mice remained high (16.2% of TCD8) (Fig. 4B). The rapid decline in the numbers of TCR-I T cells in both the spleen and the tumor was not overcome by the additional administration of FGK45 10 days after the initial adoptive transfer (Fig. 4C). Altogether, the results indicate that treatment with FGK45 leads to a transient high level accumulation of TCR-I T cells in RT4 mice.


Figure 4
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  		FIGURE 4. Conditioning of RT4 mice with FGK45 has a transient effect on the accumulation of TCR-I T cells. A and B, Groups of two to three RT4 mice of tumor-bearing age that received conditioning with FGK45 plus 1 x 106 TCR-I donor cells were analyzed 10 (A) and 20 (B) days later for the presence of TCR-I T cells in the tumors and in the spleens. Data from representative mice are shown. C, One group of RT4 mice received a second dose of FGK45 (200 µg) on day 10. Control B6 mice that received TCR-I T cells were immunized with B6/WT-19 T Ag-transformed fibroblasts to demonstrate the persistence of TCR-I T cells over time in the absence of tumor (A and B). Values in the upper right quadrant of the dot plots show the percentage of TCR-I T cells detected as a total of CD8+ cells and the average number of TCR-I T cells detected per spleen for each group (in parentheses).

 
Conditioning with FGK45 augments TCR-I T cell-mediated control of tumor progression

To determine whether the adoptive transfer of TCR-I T cells plus FGK45 had a significant effect on tumor growth and survival, groups of RT4 mice aged 5 to 5.5 mo were treated with FGK45 or rat IgG and given adoptive transfers with TCR-I T cells. A third group of RT4 mice received three treatments with TCR-I T cells plus FGK45 on days 0, 20, and 30 to determine the effect of multiple rounds of adoptive immunotherapy. By comparison with untreated RT4 mice (median age to time of death of 197 days; Fig. 5A), a single administration of TCR-I cells plus FGK45 increased the lifespan to a median age of 235 days (p = 0.0162). Survival was further increased in mice that received three rounds of immunotherapy (median age to time of death of 265 days; p = 0.0004). Although we observed an increase in lifespan after three treatments with TCR-I plus FGK45 compared with mice that received only a singe treatment, the difference between the two groups was not statistically significant (p = 0.0827). No significant increase in survival was observed for mice injected three times with FGK45 alone (median age to time of death of 201 days; p = 0.7589) or for mice given TCR-I T cells plus control rat IgG (median age to time of death of 190 days; p = 0.8324).


Figure 5
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  		FIGURE 5. Treatment with TCR-I T cells plus FGK45 reduces tumor progression in RT4 mice. Groups of RT4 mice aged 5 to 5.5 mo received adoptive transfer of 1 x 106 naive TCR-I T cells and were treated with either FGK45 or control rat IgG. A third group of RT4 mice received three treatments with TCR-I T cells plus FGK45 at days 0, 20, and 30. A fourth group of RT4 mice remained unmanipulated. A, Lifespans were monitored and plotted as the percentage of surviving animals vs age. The number of mice per group (n) and the median age of survival are indicted. Significance was determined by log rank compared with untreated RT4 mice. The bracket indicates a comparison of mice that received three doses of TCR-I T cells vs mice that received only a single dose. Asterisks indicate the curves that are significantly different from that of the untreated control group. B, Blood glucose levels in RT4 mice from A that received either a single treatment with TCR-I T cells and FGK45 or control rat IgG were measured and plotted over time beginning at 11 days before adoptive immunotherapy (day –11). Each line represents an individual mouse.

 
Additionally, we monitored the concentration of blood glucose in mice that received a single treatment with TCR-I T cells and FGK45 or control rat IgG (Fig. 5B) as an indicator of the effect on the tumor. We found that the concentration of blood glucose increased between the initiation of treatment and day 10 in RT4 mice treated with TCR-I T cells and FGK45. This was followed by a gradual decrease over the next 50 days. This observation suggests that TCR-I-based immunotherapy initially eliminated some of the insulin-producing tumor cells and is in agreement with the increased survival of this group of RT4 mice (Fig. 5A). Subsequent tumor progression after day 10 is associated with the loss of TCR-I T cells from RT4 mice between days 10 and 20 after adoptive transfer (Fig. 4). In contrast, blood glucose levels in RT4 mice treated with TCR-I T cells and control rat IgG remained stable or declined between day –1 and day 10 and is associated with lack of expansion of TCR-I T cells after such treatment (Fig. 1A). Over half of the mice in this control group died before day 60 after adoptive transfer. Taken together, these results demonstrate that the enhanced accumulation of TCR-I T cells within established tumors of RT4 mice is associated with the control of tumor progression and the prolonged survival of tumor-bearing mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The TCD8 response to cancer is often characterized by epitope multiplicity, providing several potential targets for immunotherapy (3). Effective immunotherapy may require targeting multiple epitopes to avoid Ag loss variants and to promote efficient control of tumor progression (40). One complication of this strategy is that TCD8 responding to some epitopes may be more sensitive to the effects of peripheral tolerance than others (8, 9, 41). This may be due to, among other factors, the biological properties of the epitope, the nature of the tumor or self-Ag-expressing tissue, or the sensitivity of the responding T cells to tolerance-inducing mechanisms. In this study, we demonstrate that conditioning the host with the anti-CD40 agonist Ab FGK45 can postpone the loss of TCD8 responsiveness toward an epitope that is highly tolerogenic, resulting in a delay in the progression of established tumors.

The SV40 T Ag epitope I was shown previously to form relatively long-lived complexes with H-2Db molecules on the cell surface compared with the other H-2b-restricted SV40 T Ag epitopes (27, 42). Although the naturally processed form of this peptide has not been defined, previous studies showed that the synthetic 10-mer peptide SAINNYAQKL rather than one of the 9-mer peptides binds most efficiently to H-2Db (42). The relatively high stability of epitope I/Db complexes could potentially lead to their high level accumulation at the tumor cell surface but also might play a role in sustained Ag presentation on the surface of professional APCs following cross-presentation compared with the other T Ag epitopes (43). Under noninflammatory steady-state conditions, high-level epitope presentation on professional APCs may result in tolerance induction of naive epitope I-specific TCD8 (35).

The speed with which TCR-I T cells are eliminated from RT4 mice provides a difficult challenge for the development of immunotherapeutic strategies. In the present study, the conditioning of tumor-bearing RT4 mice with FGK45 results in both an increase in the number of cells that accumulate in the lymphoid organs early after TCR-I T cell transfer and a dramatic infiltration into pancreatic tumors. The lack of infiltration of tumor-specific T lymphocytes into the tumor stroma has been viewed as a major obstacle to immunotherapeutic approaches to cancer (37, 44, 45). When T lymphocyte infiltration does occur, it has been shown to correlate with a positive disease prognosis (38, 39, 46, 47). Histological analysis of tumors has revealed that lymphocytes often fail to penetrate into the tumor stroma. Instead, T lymphocytes are detected at the tumor margin and fail to result in tumor elimination (48, 49).

Mechanisms that may limit T cell infiltration include immunologic ignorance (50), the presence of an irregular vasculature (37), and lack of proper homing receptors or chemokine production within the tumor (11). Additional treatments such as exposure to radiation or intratumoral injection of proinflammatory substances (e.g., CpGs) may be necessary to "open" the tumor stroma for efficient T cell infiltration (37, 51, 52). Our data clearly demonstrate that TCR-I T cells heavily infiltrate insulinomas and closely associate with tumor cells after the administration of FGK45, consistent with decreased tumor progression. It is not clear from the results presented here whether FGK45 conditioning of the host directly results in access to the tumor stroma or is a consequence of other events initiated following the induction of a robust immune response toward epitope I. In vivo conditioning of mice with FGK45 could potentially lead to enhanced T lymphocyte trafficking into the tissues due to the triggering of CD40 on vascular endothelial cells that results in enhanced expression of adhesion molecules and increased lymphocyte binding (53, 54).

Based on previous studies in related mouse models, the mechanism by which anti-CD40 conditioning leads to enhanced priming against the endogenous T Ag is likely through the activation of professional APCs that have taken up T Ag via cross-presentation (10, 18, 19, 55). Thus, the positive effect of anti-CD40 conditioning may be most useful for tumor Ags that are efficiently cross-presented in vivo. A similar enhancement of TCD8 accumulation toward self-Ag following anti-CD40 conditioning has been observed in several other models (14, 56). Hernandez et al. (56) found that the anti-CD40 conditioning of mice that express influenza hemagglutinin from the rat insulin promoter induces a robust accumulation of adoptively transferred hemagglutinin-specific TCR transgenic T cells in the pancreatic lymph node. However, these T cells were deficient for IFN-{gamma} production unless mice were also injected with IL-12. The authors suggest that CD40 ligation only partially substitutes for T cell help (56). We made a similar observation in RT4 mice in which a subset of tumor-infiltrating TCR-I TCD8 were able to produce IFN-{gamma}, suggesting that a proportion of the tumor-infiltrating TCR-I T cells are anergized by 10 days after adoptive transfer despite promoting a delay in tumor progression. Using a model in which the lymphocytic choriomeningitis virus glycoprotein 33 (GP33) epitope is expressed ubiquitously from the H2-Kb promoter, Pircher and colleagues (14, 57) found that adoptively transferred TCR transgenic TCD8 specific for GP33 were rapidly tolerized in vivo. However, the treatment of GP33 transgenic mice with anti-CD40 induced a massive expansion of adoptively transferred TCR transgenic T cells that resulted in the destruction of both lymphoid organs and the liver, a clear sign that these T cells had acquired effector functions. It should be noted that T cell activation in the GP33 model is not dependent on cross-presentation of the Ag, because all cells are capable of presenting the epitope directly. Together, these studies demonstrate that anti-CD40 conditioning can result in a delay in TCD8 tolerance due to an increase in the magnitude of T cell expansion following the recognition of self-Ag in vivo and the subsequent infiltration of the Ag-expressing tissues.

The TCD8-mediated reduction in tumor progression shown in the current study contrasts with the results from a previous study in which TCD8 targeting the H-2Kb-restricted epitope IV, recruited from the endogenous repertoire of RT4 mice by immunization, were able to persistently infiltrate established tumors but were unable to alter tumor progression (9). In that case, the epitope IV-specific, tumor-infiltrating TCD8 retained their effector function and were not deleted from RT4 mice. Thus, the escape of insulinomas from TCD8 specific for these two epitopes might be mediated by different mechanisms. In the case of the endogenous epitope IV-specific TCD8, escape may be controlled by changes within the tumor that protect the tumor cells from the resident effector T cells (58). In contrast, tumor escape from epitope I-specific TCD8 is based on elimination of the responding TCD8. These results indicate that TCD8-based strategies for cancer that successfully target one epitope may not apply to all tumor-associated epitopes, even within the same tumor Ag.

We also note that the effect of conditioning with FGK45 on TCD8 persistence can vary dramatically when targeting the same epitope in two different models of self-Ag expression. Previously, we found that TCR-I T cells are also suboptimally triggered against endogenous T Ag in line 501 mice (5). These mice express T Ag from the {alpha}-amylase promoter, resulting in the development of osteogenic osteosarcomas (59). After initial proliferation, adoptively transferred TCR-I T cells are eliminated from the lymphoid tissues within 3 wk (5). However, FGK45 conditioning of line 501 mice led to long-term persistence of TCR-I T cells for >1 year vs the transient effect that was observed in RT4 mice. One possible explanation for this difference is that the presence of rapidly progressing tumors in RT4 mice leads to accelerated deletion and a more immunosuppressive environment than in the more slowly developing tumor environment of line 501 mice. This explanation seems unlikely, however, because TCR-I transfer plus FGK45 conditioning of RT4 mice before tumor formation results in similar kinetics of T cell response and deletion from the peripheral lymphoid tissues (data not shown). Alternatively, this difference may be explained by the unique nature of tissues expressing T Ag. Recent evidence suggests that pancreatic islet cells may be involved in the maintenance of self-tolerance. In a mouse model of diabetes, expression of the inhibitory ligand programmed death ligand 1 (PD-L1) on the surface of pancreatic islet cells was reported to be critical for inhibiting autoreactive PD-1-expressing CD4+ T cells (60). Moreover, deletion of autoreactive TCD8 targeting an Ag expressed by the pancreatic β cells was shown to be PD-L1-dependent, although the requirement for PD-L1 expression on the pancreatic cells themselves was not demonstrated (61). Thus, one explanation for the rapid onset of tolerance in line RT4 mice despite the robust triggering following anti-CD40 conditioning is that T cell interactions with Ag presented by the pancreatic islet cells accelerates the deletion of activated T cells.

Rapid elimination of TCD8 following anti-CD40 administration has been observed previously in several systems (14, 56, 62). In particular, Kedl et al. (62) demonstrated that the administration of anti-CD40 into mice inoculated 1 wk earlier with OVA-expressing B16 melanoma cells enhanced TCD8 accumulation within the tumors, but these TCD8 were eliminated from the host after 1 week. This rapid elimination could be prevented if the mice received simultaneous immunization with a recombinant vaccinia virus expressing the tumor Ag. The authors suggested that additional inflammatory signals are required to promote a long-lived T cell response. In RT4 mice the rapid deletion of TCR-I T cells from the lymphoid tissues is unlikely to be due solely to the administration of anti-CD40, because we previously found that immunization of RT4 mice with wild-type T Ag-transformed cells the day following adoptive transfer resulted in a similar expansion and attrition from the lymphoid organs (9). Thus, in the case of RT4 mice, rapid loss of the responding TCR-I T cells appears to be an intrinsic property of the immunologic environment. A recent study by Ahonen et al. (63) showed that a combination of signaling through CD40 and TLRs induces a more potent TCD8 response than triggering either pathway independently and leads to the induction of stable T cell memory. Thus, the provision of additional inflammatory signals in RT4 mice before or following the adoptive transfer of TCR-I T cells may prolong TCD8 survival and increase the control of tumor progression.

Additional help from CD4+ T cells may be required to establish a more prolonged tumor-specific TCD8 response (56, 64, 65, 66). In a model similar to that of RT4 mice, Ganss and colleagues (36) showed that T Ag-specific CD4+ T cells can inhibit the onset of tolerance among T Ag-specific TCD8. The adoptive transfer of T Ag-specific, in vitro activated CD8+ and CD4+ TCR transgenic T cells plus the application of CpG adjuvant resulted in T cell infiltration of tumors and the long-term survival of experimental mice. These data and similar results obtained in InsHA (56) and RIP-OVA (67) mice define the need for additional help from CD4+ T cells to achieve the long-term survival of tumor-specific TCD8 in the continued presence of self-Ag. Indeed, the onset of peripheral tolerance toward epitope I is accelerated in T Ag transgenic mice in the absence of CD4+ T cells (52). Identifying which components of T cell help contribute to this enhanced survival of TCD8 will be critical for the further design of immunotherapeutic approaches.

In conclusion, the present results show that the development of peripheral tolerance can be delayed by the administration of an anti-CD40 agonist Ab, resulting in enhanced priming against the endogenous T Ag. The fact that the endogenous expression of a self-Ag can be used for the expansion of tumor-reactive T cells without any need for specific immunization following the administration of an anti-CD40 mAb might be an important consideration for therapy in humans. Efficient accumulation of functional tumor-specific TCD8 within established tumors was associated with a significant delay in tumor progression. Thus, even highly tolerogenic tumor Ags can be targeted for TCD8-mediated tumor immunotherapy.


   Acknowledgments
 
We thank Melanie Epler for technical assistance on this project. We are also grateful for the technical assistance of Nate Schafer in the Flow Cytometry Core Facility of the Pennsylvania State University College of Medicine. Microscopic analysis was performed with the help of Dr. Alistair Barber and Rhona Ellis in the Microscopy Imaging Core Facility of the Pennsylvania State University College of Medicine. We are grateful to Dr. Stephen Schoenberger (La Jolla Institute for Allergy and Immunology, La Jolla, CA) for providing the FGK45 Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 study was supported by National Cancer Institute/National Institutes of Health Grants CA-25000 (to S.S.T.) and CA-34196 (to B.B.K.); and American Cancer Society Research Scholar Grant 04-059-01-LIB (to T.D.S.). Back

2 Current address: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Back

3 Address correspondence and reprint requests to Dr. Todd D. Schell, Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail address: tschell{at}psu.edu Back

4 Abbreviations used in this paper: TCD8, CD8+T cell; DAPI, 4',6'-diamidino-2-phenylindole; Db/Flu, H-2Db/Flu NP epitope; Db/I, H-2Db/T Ag epitope I; Flu, influenza virus; GP33, glycoprotein 33; NP, nucleoprotein; PD-L1, programmed death ligand 1; RT4, RIP1-Tag4; TCR-I, TCR transgenic TCD8 specific for T Ag epitope I. Back

Received for publication April 17, 2007. Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. De Visser, K. E., T. N. Schumacher, A. M. Kruisbeek. 2003. CD8+ T cell tolerance and cancer immunotherapy. J. Immunother. 26: 1-11. [Medline]
  2. Pardoll, D.. 2003. Does the immune system see tumors as foreign or self?. Annu. Rev. Immunol. 21: 807-839. [Medline]
  3. Boon, T., P. van der Bruggen. 1996. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183: 725-729. [Free Full Text]
  4. Gattinoni, L., D. J. Powell, Jr, S. A. Rosenberg, N. P. Restifo. 2006. Adoptive immunotherapy for cancer: building on success. Nat. Rev. Immunol. 6: 383-393. [Medline]
  5. Staveley-O’Carroll, K., T. D. Schell, M. Jimenez, L. M. Mylin, M. J. Tevethia, S. P. Schoenberger, S. S. Tevethia. 2003. In vivo ligation of CD40 enhances priming against the endogenous tumor antigen and promotes CD8+ T cell effector function in SV40 T antigen transgenic mice. J. Immunol. 171: 697-707. [Abstract/Free Full Text]
  6. 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: 1178-1183. [Abstract/Free Full Text]
  7. Overwijk, W. W., M. R. Theoret, S. E. Finkelstein, D. R. Surman, L. A. de Jong, F. A. Vyth-Dreese, T. A. Dellemijn, P. A. Antony, P. J. Spiess, D. C. Palmer, et al 2003. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J. Exp. Med. 198: 569-580. [Abstract/Free Full Text]
  8. 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: 3002-3012. [Abstract/Free Full Text]
  9. Otahal, P., T. D. Schell, S. C. Hutchinson, B. B. Knowles, S. S. Tevethia. 2006. Early immunization induces persistent tumor-infiltrating CD8+ T cells against an immunodominant epitope and promotes lifelong control of pancreatic tumor progression in SV40 tumor antigen transgenic mice. J. Immunol. 177: 3089-3099. [Abstract/Free Full Text]
  10. Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19: 47-64. [Medline]
  11. Gajewski, T. F., Y. Meng, C. Blank, I. Brown, A. Kacha, J. Kline, H. Harlin. 2006. Immune resistance orchestrated by the tumor microenvironment. Immunol. Rev. 213: 131-145. [Medline]
  12. 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: 780-787. [Medline]
  13. 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: 774-779. [Medline]
  14. Roth, E., J. Schwartzkopff, H. Pircher. 2002. CD40 ligation in the presence of self-reactive CD8 T cells leads to severe immunopathology. J. Immunol. 168: 5124-5129. [Abstract/Free Full Text]
  15. Garza, K. M., S. M. Chan, R. Suri, L. T. Nguyen, B. Odermatt, S. P. Schoenberger, P. S. Ohashi. 2000. Role of antigen-presenting cells in mediating tolerance and autoimmunity. J. Exp. Med. 191: 2021-2027. [Medline]
  16. Maxwell, J. R., J. D. Campbell, C. H. Kim, A. T. Vella. 1999. CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion. J. Immunol. 162: 2024-2034. [Abstract/Free Full Text]
  17. Quezada, S. A., L. Z. Jarvinen, E. F. Lind, R. J. Noelle. 2004. CD40/CD154 interactions at the interface of tolerance and immunity. Annu. Rev. Immunol. 22: 307-328. [Medline]
  18. 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: 478-480. [Medline]
  19. 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: 480-483. [Medline]
  20. van Mierlo, G. J., Z. F. Boonman, H. M. Dumortier, A. T. den Boer, M. F. Fransen, J. Nouta, E. I. van der Voort, R. Offringa, R. E. Toes, C. J. Melief. 2004. Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication. J. Immunol. 173: 6753-6759. [Abstract/Free Full Text]
  21. Van Mierlo, G. J., A. T. den Boer, J. P. Medema, E. I. van der Voort, M. F. Fransen, R. Offringa, C. J. Melief, R. E. Toes. 2002. CD40 stimulation leads to effective therapy of CD40– tumors through induction of strong systemic cytotoxic T lymphocyte immunity. Proc. Natl. Acad. Sci. USA 99: 5561-5566. [Abstract/Free Full Text]
  22. Nguyen, L. T., A. R. Elford, K. Murakami, K. M. Garza, S. P. Schoenberger, B. Odermatt, D. E. Speiser, P. S. Ohashi. 2002. Tumor growth enhances cross-presentation leading to limited T cell activation without tolerance. J. Exp. Med. 195: 423-435. [Abstract/Free Full Text]
  23. Murphy, W. J., L. Welniak, T. Back, J. Hixon, J. Subleski, N. Seki, J. M. Wigginton, S. E. Wilson, B. R. Blazar, A. M. Malyguine, et al 2003. Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses. J. Immunol. 170: 2727-2733. [Abstract/Free Full Text]
  24. Hanahan, D.. 1985. Heritable formation of pancreatic β-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315: 115-122. [Medline]
  25. 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: 3916-3920. [Abstract/Free Full Text]
  26. Tevethia, S. S., T. D. Schell. 2002. The immune response to SV40, JCV and BKV. K. Khalili, Jr, and G. L. Stoner, Jr, eds. Human Polyomaviruses: Molecular and Clinical Perspectives 585-610. Wiley-Liss, New York.
  27. Mylin, L. M., R. H. Bonneau, J. D. Lippolis, S. S. Tevethia. 1995. Hierarchy among multiple H-2b-restricted cytotoxic T lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69: 6665-6677. [Abstract]
  28. Mylin, L. M., T. D. Schell, D. Roberts, M. Epler, A. Boesteanu, E. J. Collins, J. A. Frelinger, S. Joyce, S. S. Tevethia. 2000. Quantitation of CD8+ T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes. J. Virol. 74: 6922-6934. [Abstract/Free Full Text]
  29. Tanaka, Y., R. W. Anderson, W. L. Maloy, S. S. Tevethia. 1989. Localization of an immunorecessive epitope on SV40 T antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide. Virology 171: 205-213. [Medline]
  30. Schaefer, B. C., M. L. Schaefer, J. W. Kappler, P. Marrack, R. M. Kedl. 2001. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214: 110-122. [Medline]
  31. Pretell, J., R. S. Greenfield, S. S. Tevethia. 1979. Biology of simian virus 40 (SV40) transplantation rejection antigen (TrAg): in vitro demonstration of SV40 TrAg in SV40 infected non-permissive mouse cells by the lymphocyte-mediated cytotoxicity assay. Virology 97: 32-41. [Medline]
  32. Rolink, A., F. Melchers, J. Andersson. 1996. The SCID but not the RAG-2 gene product is required for Sµ-S{epsilon} heavy chain class switching. Immunity 5: 319-330. [Medline]
  33. Skinner, P. J., M. A. Daniels, C. S. Schmidt, S. C. Jameson, A. T. Haase. 2000. Cutting edge: in situ tetramer staining of antigen-specific T cells in tissues. J. Immunol. 165: 613-617. [Abstract/Free Full Text]
  34. Cavender, J. F., A. Conn, M. Epler, H. Lacko, M. J. Tevethia. 1995. Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts. J. Virol. 69: 923-934. [Abstract]
  35. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch, R. M. Steinman, M. C. Nussenzweig. 2001. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194: 769-779. [Abstract/Free Full Text]
  36. Garbi, N., B. Arnold, S. Gordon, G. J. Hammerling, R. Ganss. 2004. CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction. J. Immunol. 172: 5861-5869. [Abstract/Free Full Text]
  37. Ganss, R., E. Ryschich, E. Klar, B. Arnold, G. J. Hammerling. 2002. Combination of T-cell therapy and trigger of inflammation induces remodeling of the vasculature and tumor eradication. Cancer Res. 62: 1462-1470. [Abstract/Free Full Text]
  38. Galon, J., A. Costes, F. Sanchez-Cabo, A. Kirilovsky, B. Mlecnik, C. Lagorce-Pages, M. Tosolini, M. Camus, A. Berger, P. Wind, et al 2006. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313: 1960-1964. [Abstract/Free Full Text]
  39. Prall, F., T. Duhrkop, V. Weirich, C. Ostwald, P. Lenz, H. Nizze, M. Barten. 2004. Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability. Hum. Pathol. 35: 808-816. [Medline]
  40. Yee, C., J. A. Thompson, D. Byrd, S. R. Riddell, P. Roche, E. Celis, P. D. Greenberg. 2002. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc. Natl. Acad. Sci. USA 99: 16168-16173. [Abstract/Free Full Text]
  41. Slifka, M. K., J. N. Blattman, D. J. Sourdive, F. Liu, D. L. Huffman, T. Wolfe, A. Hughes, M. B. Oldstone, R. Ahmed, M. G. Von Herrath. 2003. Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy. J. Immunol. 170: 1231-1239. [Abstract/Free Full Text]
  42. Lippolis, J. D., L. M. Mylin, D. T. Simmons, S. S. Tevethia. 1995. Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen. J. Virol. 69: 3134-3146. [Abstract]
  43. Otahal, P., S. C. Hutchinson, L. M. Mylin, M. J. Tevethia, S. S. Tevethia, T. D. Schell. 2005. Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope. J. Immunol. 175: 700-712. [Abstract/Free Full Text]
  44. Singh, S., S. R. Ross, M. Acena, D. A. Rowley, H. Schreiber. 1992. Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J. Exp. Med. 175: 139-146. [Abstract/Free Full Text]
  45. Yu, P., D. A. Rowley, Y. X. Fu, H. Schreiber. 2006. The role of stroma in immune recognition and destruction of well-established solid tumors. Curr. Opin. Immunol. 18: 226-231. [Medline]
  46. Elder, D.. 1999. Tumor progression, early diagnosis and prognosis of melanoma. Acta Oncol. 38: 535-547. [Medline]
  47. Zhang, L., J. R. Conejo-Garcia, D. Katsaros, P. A. Gimotty, M. Massobrio, G. Regnani, A. Makrigiannakis, H. Gray, K. Schlienger, M. N. Liebman, et al 2003. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N. Engl. J. Med. 348: 203-213. [Abstract/Free Full Text]
  48. Ibe, S., Z. Qin, T. Schuler, S. Preiss, T. Blankenstein. 2001. Tumor rejection by disturbing tumor stroma cell interactions. J. Exp. Med. 194: 1549-1559. [Abstract/Free Full Text]
  49. Blankenstein, T.. 2005. The role of tumor stroma in the interaction between tumor and immune system. Curr. Opin. Immunol. 17: 180-186. [Medline]
  50. 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: 1058-1064. [Medline]
  51. Yu, P., Y. Lee, W. Liu, R. K. Chin, J. Wang, Y. Wang, A. Schietinger, M. Philip, H. Schreiber, Y. X. Fu. 2004. Priming of naive T cells inside tumors leads to eradication of established tumors. Nat. Immunol. 5: 141-149. [Medline]
  52. Schell, T. D., S. S. Tevethia. 2001. Control of advanced choroid plexus tumors in SV40 T antigen transgenic mice following priming of donor CD8+ T lymphocytes by the endogenous tumor antigen. J. Immunol. 167: 6947-6956. [Abstract/Free Full Text]
  53. Hollenbaugh, D., N. Mischel-Petty, C. P. Edwards, J. C. Simon, R. W. Denfeld, P. A. Kiener, A. Aruffo. 1995. Expression of functional CD40 by vascular endothelial cells. J. Exp. Med. 182: 33-40. [Abstract/Free Full Text]
  54. Karmann, K., C. C. Hughes, J. Schechner, W. C. Fanslow, J. S. Pober. 1995. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc. Natl. Acad. Sci. USA 92: 4342-4346. [Abstract/Free Full Text]
  55. Prilliman, K. R., E. E. Lemmens, G. Palioungas, T. G. Wolfe, J. P. Allison, A. H. Sharpe, S. P. Schoenberger. 2002. Cutting edge: a crucial role for B7-CD28 in transmitting T help from APC to CTL. J. Immunol. 169: 4094-4097. [Abstract/Free Full Text]
  56. Hernandez, J., S. Aung, K. Marquardt, L. A. Sherman. 2002. Uncoupling of proliferative potential and gain of effector function by CD8+ T cells responding to self-antigens. J. Exp. Med. 196: 323-333. [Abstract/Free Full Text]
  57. Roth, E., H. Pircher. 2004. IFN-{gamma} promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells. J. Immunol. 172: 1588-1594. [Abstract/Free Full Text]
  58. Ye, X., A. Kralli, R. Ge, R. P. Ricciardi, B. B. Knowles. 1994. Down-regulation of MHC class I antigen in insulinoma cells controlled by the R1 element of the H-2 enhancer. Oncogene 9: 1195-1204. [Medline]
  59. 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: 115-125. [Medline]
  60. Keir, M. E., S. C. Liang, I. Guleria, Y. E. Latchman, A. Qipo, L. A. Albacker, M. Koulmanda, G. J. Freeman, M. H. Sayegh, A. H. Sharpe. 2006. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J. Exp. Med. 203: 883-895. [Abstract/Free Full Text]
  61. Martin-Orozco, N., Y. H. Wang, H. Yagita, C. Dong. 2006. Cutting edge: programed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens. J. Immunol. 177: 8291-8295. [Abstract/Free Full Text]
  62. Kedl, R. M., M. Jordan, T. Potter, J. Kappler, P. Marrack, S. Dow. 2001. CD40 stimulation accelerates deletion of tumor-specific CD8+ T cells in the absence of tumor-antigen vaccination. Proc. Natl. Acad. Sci. USA 98: 10811-10816. [Abstract/Free Full Text]
  63. Ahonen, C. L., C. L. Doxsee, S. M. McGurran, T. R. Riter, W. F. Wade, R. J. Barth, J. P. Vasilakos, R. J. Noelle, R. M. Kedl. 2004. Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN. J. Exp. Med. 199: 775-784. [Abstract/Free Full Text]
  64. Bachmann, M. F., K. Schwarz, P. Wolint, E. Meijerink, S. Martin, V. Manolova, A. Oxenius. 2004. Cutting edge: distinct roles for T help and CD40/CD40 ligand in regulating differentiation of proliferation-competent memory CD8+ T cells. J. Immunol. 173: 2217-2221. [Abstract/Free Full Text]
  65. Montfort, M. J., H. G. Bouwer, C. R. Wagner, D. J. Hinrichs. 2004. The development of functional CD8 T cell memory after Listeria monocytogenes infection is not dependent on CD40. J. Immunol. 173: 4084-4090. [Abstract/Free Full Text]
  66. Behrens, G. M., M. Li, G. M. Davey, J. Allison, R. A. Flavell, F. R. Carbone, W. R. Heath. 2004. Helper requirements for generation of effector CTL to islet β cell antigens. J. Immunol. 172: 5420-5426. [Abstract/Free Full Text]
  67. Belz, G. T., G. M. Behrens, C. M. Smith, J. F. Miller, C. Jones, K. Lejon, C. G. Fathman, S. N. Mueller, K. Shortman, F. R. Carbone, W. R. Heath. 2002. The CD8{alpha}+ dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196: 1099-1104. [Abstract/Free Full Text]



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A. M. Tatum, L. M. Mylin, S. J. Bender, M. A. Fischer, B. A. Vigliotti, M. J. Tevethia, S. S. Tevethia, and T. D. Schell
CD8+ T Cells Targeting a Single Immunodominant Epitope are Sufficient for Elimination of Established SV40 T Antigen-Induced Brain Tumors
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