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The Journal of Immunology, 2004, 172: 6074-6079.
Copyright © 2004 by The American Association of Immunologists

The Tumoricidal Activity of Memory CD8+ T Cells Is Hampered by Persistent Systemic Antigen, but Full Functional Capacity Is Regained in an Antigen-Free Environment1

Annemieke Th. den Boer*, Geertje J. D. van Mierlo*, Marieke F. Fransen*, Cornelis J. M. Melief*, Rienk Offringa* and René E. M. Toes2,{dagger}

Departments of * Immunohematology and Blood Transfusion, and {dagger} Rheumatology, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Naive T cells can be tolerized in the periphery by diverse mechanisms. However, the extent to which memory T cells are susceptible to tolerance induction is less well defined. Vaccination of mice with a minimal CTL epitope derived from human adenovirus type 5 E1A in IFA s.c. readily tolerizes naive as well as recently activated CD8+ T cells due to the overwhelming systemic and persistent presence of the peptide. We have now studied the effect of this peptide on established memory cells, which were induced at least 50 days before by virus vaccination. Memory cells did not undergo peripheral deletion and kept their ability to produce IFN-{gamma} as well as their cytolytic activity in response to Ag directly ex vivo. However, memory CTL responses in virus vaccinated mice injected with peptide ceased to control tumor outgrowth. Interestingly, functional capacities were regained when T cells were transferred to an Ag-free environment in vivo as determined by their ability to reject an otherwise lethal tumor challenge. Together, these findings indicate that memory CTL responses can be functionally incapacitated, but are not, in contrast to naive or recently activated T cells, irreversibly tolerized by persistent systemic Ag, as memory T cells quickly regain effector function upon disappearance of the Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To guard the integrity of the body, the immune system has to eliminate pathogens while preventing reactivity against self-Ags. In the thymus, developing T cells with high avidity for self-peptide-MHC complexes are eliminated (1). Furthermore, in the periphery several tolerizing mechanisms exist to eliminate autoreactive responses (2), for instance, as shown by studies in mice expressing a transgenic self-Ag in pancreatic {beta} cells (3). This Ag is cross-presented in the draining lymph nodes and naive TCR transgenic (tg)3 CD8+ T cells specific for the self-Ag are tolerized upon adoptive transfer. This cross-tolerization only required Ag recognition on bone marrow-derived APC. Upon injection of OVA-specific TCRtg CD4+ T cells in this system, CD8+ T cells are no longer tolerized, but cause autoimmunity. CD4+ T cells are thought to activate the cross-presenting APC endowing it with the capacity to induce a proper CD8+ T cell response. These observations indicate that the activation status of the APC is a crucial factor in the balance between immunity and tolerance of CD8+ T cells.

Another aspect that contributes to induction of tolerance or immunity is the Ag itself. For example, lymphocytic choriomeningitis virus (LCMV) strains that cause rapid and overwhelming infections in the lymphoid organs prime virus-specific T cells, which are subsequently tolerized by "exhaustion" (4). In contrast, slowly replicating LCMV strains induce long-lasting immunity in the presence of CD4+ T cell help (5). Likewise, repetitive, systemic administration of peptides results in peripheral deletion of naive CD8+ T cells, whereas s.c. injection of these peptides induces specific CTL immunity (6). Thus, T cell tolerance is favored by the absence of proper costimulation, particularly in the presence of persistent systemic Ag. In contrast, local Ag presentation combined with proper costimulation leads to T cell immunity.

Apart from the distribution kinetics and the environment in which an Ag is presented, the differentiation status of the T cells greatly influences the outcome of Ag recognition. Although memory T cells are generally thought to be less susceptible to tolerance induction than naive T cells (7), only few studies have addressed the question of susceptibility to tolerance induction of memory CD8+ T cells. For example, male B cells injected i.v. in female mice induced H-Y specific tolerance of naive T cells, whereas memory T cells against H-Y were reactivated (8). B cells are known to provide less T cell costimulatory signals than professional APCs such as dendritic cells (9). Because memory T cells are less dependent on costimulatory stimuli (10), the absence of costimulation, which tolerizes naive T cells, may not affect memory T cells. On the contrary, influenza-specific CD8+ T cells, activated 3 wk before by vaccination with influenza virus, underwent peripheral tolerance after i.v. injection of a high dose of influenza peptide (11), indicating that activated CD8+ T cells are still prone to tolerance induction. In conclusion, it remains controversial whether and in particular how memory CD8+ T cells can be tolerized.

Better understanding of the susceptibility of memory T cells to tolerance induction is important to improve vaccination strategies against diseases like cancer or the treatment of autoimmunity. Because tumors often arise from and masquerade as normal tissue cells, tumor Ags are likely to be presented to the immune system in a tolerizing manner (12). If memory T cells can be tolerized, this might affect the maintenance of long-lived T cell immunity to tumor Ags.

To further address this question, we have studied the effect of a tolerizing vaccine on memory T cells in a well-established mouse model. In earlier studies we showed that vaccination with a minimal CTL epitope derived from human adenovirus type 5 (Ad5) early region 1A (E1A) oncoprotein readily tolerizes naive E1A-specific CD8+ T cells when injected s.c. at low doses (13). The minimal level of peptide required for tolerization was 10 µg/mouse (13). After injection in IFA, the peptide spreads quickly out the IFA depot and is presented everywhere in the periphery (14, 15), most likely in the absence of sufficient costimulation, causing naive CD8+ T cell tolerance. When presented on activated dendritic cells, this peptide induces immunity (16). Furthermore, when the tolerizing E1A-peptide vaccine is combined with injection of an activating Ab against CD40, tolerance induction is initially reverted into strong priming (14). However, the primed E1A-specific T cells are finally tolerized (17). Also in this case, tolerization was strongly associated with the persistent and systemic presence of the peptide. These findings indicate that effector CD8+ T cells can still be tolerized.

We have now investigated the effect of injection of the E1A-peptide in IFA s.c. on established memory T cells that were induced at least 50 days before by virus vaccination. Memory E1A-specific T cells remained present but were functionally impaired in the presence of the persistent, systemic Ag. Interestingly, functional capacity was regained in an Ag-free environment. These findings indicate that memory CTL responses can be incapacitated by the presence of systemic and persistent Ag, but that the effector functions of these CTL are not permanently lost as they quickly regain function in the absence of Ag.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

All in vitro cultures and assays were performed in IMDM (Life Technologies, Gaithersburg, MD) supplemented with 8% FCS, 50 µM 2-ME, glutamine, and penicillin.

Mice

C57BL/6 (H-2b) mice were purchased from IFFA Credo (Paris, France). Strain 42 mice are TCRtg mice expressing the TCR {alpha}-chain and {beta}-chain derived from the H-2Db-restricted, Ad5E1A234–243-specific CTL clone 5 (13, 18).

Vaccinations

Ad5E1A234–243 (sequence SGPSNTPPEI; E1A peptide) was dissolved in DMSO and diluted in PBS. For peptide vaccination, mice were injected s.c. with peptide (20 µg) diluted in 100 µl of PBS mixed with 100 µl of IFA. Temperature-sensitive mutant Ad5ts125 (1 x 108 PFU) was injected i.m. in the hind leg.

Tumor challenge

In tumor challenge experiments, C57BL/6 mouse embryo cells, which express Ad5E1A and EJras, were collected and washed in PBS. A total of 107 cells were injected s.c. into C57BL/6 mice. Tumor volumes were measured with a caliper. Mice were sacrificed when their tumors grew larger than 1000 mm3.

Flow cytometry analysis

PE-conjugated E1A234–243-loaded H-2Db tetramers were prepared as described (19, 20) with the described modifications. During the refolding and subsequent purification steps, a mixture of protease inhibitors (Boehringer Mannheim, Mannheim, Germany) was added. BSA and glycerol were added to final concentrations of 0.5% and 16%, respectively. Tetramers were aliquoted, stored frozen, and used at a final concentration of 5–10 µg/ml. Tetramer staining of heparinized blood samples was performed after erythrocyte lysis. Directly conjugated APC-labeled mAbs against CD8 and PE-conjugated mAb against IFN-{gamma} were used (BD PharMingen, San Diego, CA). Intracellular IFN-{gamma} staining of blood samples was performed after erythrocyte lysis according to the protocol of the cytoperm/cytofix plus kit (BD PharMingen). Data acquisition and analysis were performed on a FACScan (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences).

CFSE labeling and proliferation assay

Spleen and lymph node cells from E1A-specific TCRtg strain 42 mice were isolated. Erythrocytes were depleted by ammonium chloride treatment. Spleen and lymph node cells were washed in cold PBS and incubated with 0.5 µM CFSE (Molecular Probes, Eugene, OR) in PBS at 37°C for 30 min; 5% FCS was added, and the cells were washed twice in PBS. Cell suspensions containing 4 x 106 E1A-specific CD8+ T cells were injected i.v. Three days later, spleen and lymph node cells were analyzed for the presence of CFSE-labeled CD8+ strain 42 cells by FACS analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E1A-specific memory CD8+ T cells are not deleted by vaccination with a peptide that tolerizes naive T cells

Injection of the Ad5E1A234–243-peptide in IFA s.c. in mice readily tolerizes the naive E1A-specific T cells because subsequent challenge with E1A+ tumor cells results in the inability to reject the lethal tumor challenge (13). When combined with CD40 ligation, E1A-specific tolerance is reverted into priming (14). However, the persistent systemic presence of the peptide eventually tolerizes the effector T cells, resulting in tumor outgrowth (17). To determine the effect of this tolerizing peptide vaccination on established memory CD8+ T cells, mice were vaccinated with adenovirus to induce a strong, tumor-protective E1A-specific CD8+ T cell response. The mice were left untreated for at least 50 days to allow proper memory formation. Subsequently, the mice were injected s.c. with E1A peptide in IFA. To analyze the effect of peptide treatment on the presence of E1A-specific memory CTL, the E1A-specific CTL response was followed by flow cytometric analyses using Db-E1A-tetrameric complexes.

The E1A-specific CD8+ T cells completely disappeared from the blood 4 days after peptide injection (Fig. 1). However, T cells reappeared in the blood and remained present from day 10 onward. In some mice the percentage of E1A-specific T cells in blood increased compared with the levels before peptide vaccination, whereas in other mice percentages returned to the levels before peptide injection. E1A-specific T cells did not disappear from the blood (Fig. 1), lymphoid, or nonlymphoid organs (data not shown) after peptide vaccination, but remained present even 150 days after peptide vaccination at levels similar to those in mice vaccinated with virus only. In naive mice, no CD8+ T cells can be detected that interact with the E1A tetramers (data not shown and Ref. 14). These results clearly show that memory T cells are not deleted by the peptide vaccine.



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  		FIGURE 1. Memory E1A-specific CD8+ T cells remain present after vaccination with a peptide that tolerizes naive T cells. C57BL/6 mice were injected with 1 x 108 PFU Ad5ts125 i.m. (A) or 1 x 108 PFU Ad5ts125 i.m. (B) and 55 days later with 20 µg of E1A peptide in IFA s.c. At several time points after the vaccination(s) the percentage of CD8+ T cells capable of interacting with H-2Db-E1A234–243 in blood was determined by FACS analysis. Each line represents an individual mouse. Data are representative of five independent experiments.

 
Memory E1A-specific CD8+ T cells maintain their functional capacities

E1A-specific memory T cells remained present after peptide injection as shown by flow cytometric analyses using Db-E1A-tetrameric complexes. To examine whether these memory T cells also retained their functional integrity, the ability of the E1A-specific T cells to produce IFN-{gamma} and lyse target cells in response to the specific Ag was determined directly ex vivo. E1A-specific T cells from vaccinated mice that were injected with peptide 62 days later produced IFN-{gamma} upon stimulation in vitro (Fig. 2). Furthermore, T cells obtained from virus/peptide-vaccinated animals expanded after in vitro restimulation with E1A-expressing tumor cells and were able to specifically kill target cells (data not shown). These results show that the T cells were able to respond properly to Ag exposure directly ex vivo as well as after in vitro restimulation. Together, these data indicate that the E1A-specific memory CTL remain present in normal numbers without losing their ability to display effector functions when exposed to E1A peptide.



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  		FIGURE 2. Memory E1A-specific CD8+ T cells maintain their capacity to produce IFN-{gamma} directly ex vivo. C57BL/6 mice were injected with 1 x 108 PFU Ad5ts125 i.m. or 1 x 108 PFU Ad5ts125 i.m. and 62 days later with 20 µg of E1A peptide in IFA s.c. Following 99 days after virus vaccination, the percentage of CD8+ T cells in blood capable of interacting with H-2Db-E1A234–243 or producing IFN-{gamma} was determined by FACS analysis. Numbers inset in bold represent the percentage of tetramer-positive or IFN-{gamma}-producing cells in the CD8-positive population. Data are representative of at least 20 analyses at different time points after peptide injection.

 
E1A peptide is not cleared from immune mice

Our results so far show that peptide injection in virus-vaccinated mice does not lead to deletion of E1A-specific memory T cells and that these memory T cells remain functional as analyzed directly ex vivo. Although the peptide is known to spread rapidly throughout the body and to persist for extremely long periods upon s.c. injection in IFA in naive mice (17), in virus-vaccinated mice, the memory E1A-specific T cells may rapidly clear the peptide, thereby avoiding the tolerizing effect of the persistent systemic Ag presentation. To evaluate whether the peptide persisted in the immune mice, we made use of the CFSE labeling technique (21) and E1A-specific TCRtg CD8+ T cells. These TCRtg cells only divide upon encounter with their specific Ag, the E1A peptide (data not shown and Ref. 17). Peptide was injected in virus-vaccinated mice 70 days after virus vaccination. Following 168 days after peptide injection, CFSE-labeled TCRtg cells were injected to determine whether the E1A peptide was still present. Three days later division of CFSE-labeled cells was determined by flow cytometric analysis. In virus/peptide-vaccinated mice, division of TCRtg cells was detected (Fig. 3B), indicating that the E1A peptide was still presented to the T cells 168 days after injection. In contrast, in virus-vaccinated mice without peptide immunization no division was observed. Together, these data indicate that the peptide was apparently not cleared, whereas E1A-specific T cells retained their functional capacities. Furthermore, we did not detect any macroscopical signs of autoimmunity or immunopathology in the virus-immunized mice injected with the E1A peptide or in the virus-immunized mice without peptide. Thus, these results show that the E1A peptide persists in virus-vaccinated mice and is presented to E1A-specific CTL, without induction of overt signs of autoimmunity.



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  		FIGURE 3. E1A peptide is not cleared from immune mice. A, C57BL/6 mice were left untreated (filled histogram) or injected with 20 µg of E1A peptide in IFA s.c. (line). B, C57BL/6 mice were injected with 1 x 108 PFU of Ad5ts125 i.m. (filled histogram) or with both 1 x 108 PFU of Ad5ts125 i.m. and 70 days later with 20 µg of E1A peptide in IFA s.c. (line). At 168 days after peptide vaccination, CFSE-labeled cells from E1A-specific TCRtg mice were injected i.v. Three days later, inguinal lymph node cells from the peptide-draining site were tested for the presence of the E1A peptide by measuring E1A-specific cell division of CD8+ CFSE-labeled cells by FACS analysis. Histograms show CFSE label gated on living CD8+ cells.

 
Delayed tumor outgrowth in immune mice injected with E1A peptide

Because no indications were obtained of CTL-mediated (auto) immune-pathology toward the systemically and persistently presented E1A peptide, despite the presence of E1A-specific memory CTL in virus-vaccinated mice, we examined whether E1A-specific T cells in virus/peptide-vaccinated mice retained their functional capacities in vivo by examining their ability to clear E1A+ tumor cells. E1A-specific CD8+ T cell immunity is crucial for eradication of these tumor cells (13). Virus-vaccinated mice were injected with the E1A peptide after 85 days. Twenty-nine days after peptide injection a tumor challenge was given. Virus-vaccinated mice injected with the E1A peptide were able to slow down tumor outgrowth as compared with nonvaccinated mice, but eventually all peptide-injected mice developed a lethal tumor (Fig. 4). In contrast, vaccination with virus alone completely protected all mice against tumor outgrowth. Thus, although the E1A-specific T cells in virus-vaccinated mice injected with the peptide remained present without losing their functional capacities when analyzed directly ex vivo, they are apparently not able to control tumor outgrowth in vivo in an environment in which the peptide is persistently and systemically presented.



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  		FIGURE 4. E1A+ tumors grow out in immune mice injected with E1A peptide. C57BL/6 mice (n = 4) were injected with 1 x 108 PFU Ad5ts125 i.m. ({circ}), and (n = 6) 1 x 108 PFU Ad5ts125 i.m. and 85 days later with 20 µg of E1A peptide in IFA s.c. (•), or (n = 4) left untreated ({square}). At 114 days after virus vaccination, mice were challenged with 1 x 107 live Ad5E1/ras cells in the flank s.c. The percentage of surviving mice is shown. Significant difference shown in untreated vs Ad5ts125, p < 0.01; untreated vs Ad5ts125 plus peptide, p < 0.002; Ad5ts125 vs Ad5ts125 plus peptide, p < 0.01. Data are representative of two independent experiments.

 
Adoptively transferred CD8+ T cells from virus-vaccinated mice injected with the E1A peptide regain their functional capacities in vivo when the Ag is no longer present

Although the E1A-specific memory T cells from virus-vaccinated mice injected with peptide do not lose their functional abilities when analyzed in vitro, their functional capacity in vivo is apparently limited as indicated by the hampered ability to clear E1A-expressing tumors and the absence of autoimmunity. To study whether the persistent presence of the peptide influenced the functional capacity of the E1A-specific T cells, we isolated spleen cells from virus/peptide-vaccinated mice and adoptively transferred the cells, after enrichment for CD8+ T cells, to naive mice or mice injected with the peptide in IFA. Subsequently, these mice were given a tumor challenge. In the presence of the peptide, transferred E1A-specific T cells were not able to control tumor outgrowth (Fig. 5). However, after transfer of the E1A-specific T cells to an Ag-free animal, tumor outgrowth was prevented. Adoptive transfer of similar amounts of naive E1A-specific T cells, isolated from TCRtg mice, into Ag-free animals are unable to clear a subsequent tumor challenge (G.J.D. van Mierlo, unpublished observations).



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  		FIGURE 5. Adoptively transferred E1A+ T cells from virus/peptide-vaccinated mice regain functional activity in peptide-free environment. Spleen and lymph node cells from mice vaccinated with 1 x 108 PFU Ad5ts125 combined with 20 µg of E1A peptide in IFA s.c. were enriched for T cells by erythrocyte lysis and nylon wool passage and transferred to naive C57BL/6 mice ({circ}) or C57BL/6 mice that were injected with 20 µg of E1A peptide in IFA s.c. on the same day (•). Two days later mice were challenged with 1 x 107 live Ad5E1/ras cells in the flank. The percentage of surviving mice is shown. Data are representative of two independent experiments.

 
Together, these results indicate that memory CTL effector function is blunted in the presence of systemic, persistent Ag. Memory CTL differ from recently activated CD8+ T cells because they remain present in normal numbers in vivo and preserve their functional capacities ex vivo. Full in vivo function is regained upon transfer to a systemic Ag-free environment, indicating that the presence of Ag dictates the outcome of memory CTL responsiveness.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that established memory CD8+ T cells can be affected by a persistently and systemically presented peptide. In contrast to naive and effector CD8+ T cells, memory T cells are not tolerized after peptide injection; they remain present in normal numbers and display effector functions directly ex vivo. However, the memory CD8+ T cells are functionally compromised in vivo in the presence of the peptide, as shown by the outgrowth of tumors. Interestingly, upon transfer to an Ag-free environment, memory T cells regain their effectiveness. Thus, a clear difference between naive, effector, and memory T cells is observed in susceptibility to a ‘tolerizing’ environment characterized by the systemic and persistent presence of Ag.

The mechanisms underlying these differences have not yet been resolved. However, several parameters may contribute to the increased resistance of memory CD8+ T cells to tolerance induction. For activation of naive and memory T cells, different costimulatory requirements prevail (10). After s.c. injection in IFA, the E1A peptide is distributed quickly throughout the mouse (14, 15), most likely resulting in Ag presentation without costimulation. Naive E1A-specific CD8+ T cells are tolerized after peptide injection, which is in line with the requirement for costimulation of naive T cell priming. Indeed, coinjection of activating Abs against CD40 or poly(I:C) results in T cell priming, most likely because these agents activate dendritic cells and thereby enable them to provide the necessary costimulation. Memory T cells are thought to be less dependent on costimulatory signals (7, 22), which probably contributes to the fact that E1A-specific memory T cells, in contrast to naive T cells, remain present, and in some mice, expand upon peptide vaccination. Furthermore, memory T cells contain elevated levels of antiapoptotic molecules, like Bcl-2 (23). Constitutive expression of Bcl-xL or Bcl-2 prevents peptide-induced T cell deletion (24). Therefore, elevated antiapoptotic molecules may result in an increased resistance of established memory T cells to apoptosis induction under tolerizing circumstances.

Although memory T cells are more resistant to tolerance induction by encounter of overwhelming Ag, the memory response is impaired in the presence of the peptide as shown by the final outgrowth of tumors. These results are very intriguing. The exact reason why memory T cells are ultimately unable to resist a tumor challenge in the presence of their cognate peptide remains to be elucidated. The persistent Ag depot may distract memory T cells from homing to the tumor, resulting in too low numbers of effector T cells at the tumor site to effectively control the rapidly expanding tumor cells. However, this does not seem likely as i.v. injected peptide-coated target cells are also less efficiently killed in ‘memory’ mice that received peptide (data not shown). Conversely, the persistent encounter with Ag everywhere may affect robust effector function of the memory T cells. For optimal cytolytic capacity of T cells, release of cytolytic granules following direction of the TCRs and other molecules toward the target cell presenting the specific MHC-peptide complex is crucial (25, 26). In an environment characterized by the ubiquitous presence of the peptide, this directed exertion of effector function may be hampered. Moreover, continuous triggering of the TCR may lead to the expression of molecules that inhibit T cell function. Recently, Stamou et al. (27) have shown that naive CD8+ TCRtg T cells that are chronically exposed to their cognate Ag become unresponsive to the peptide in vitro. CD5, a negative regulator of T cell signaling, was highly expressed on these anergic T cells. Transfer to an Ag-free environment resulted in regain of functional capacity. This was associated with a return of CD5 expression to normal levels. We could not detect increased CD5 expression on E1A-specific memory CD8+ T cells that were chronically exposed to the E1A peptide (data not shown), indicating that this negative regulator is most likely not involved in the reduced functional capacity of the E1A-specific memory T cells. Clearly, further investigations are needed to elucidate the mechanism behind the reduced tumoricidal effector function of the memory CD8+ T cells in the presence of persistent systemic Ag.

In contrast to our observations, a recent report described the tolerization of CD8+ T cells by high-dose peptide injection 3 wk after virus vaccination (11). In our study, peptide was injected at least 50 days after virus vaccination to establish memory formation. A difference in susceptibility to tolerance induction between effector and established memory T cells may explain the disparity between these two models. Kaech et al. (28) have shown that memory characteristics are gradually gained between day 8 and day 40 after LCMV infection. Likewise, increased resistance of memory T cells to tolerance induction may gradually develop over time.

In conclusion, we have shown that established memory CD8+ T cells are more resistant than naive and effector T cells to tolerance induction by persistent and systemic Ag encounter. However, systemic and persistent encounter of Ag results in diminished/blunted effector function of established memory CD8+ T cells and final incompetence to control tumor outgrowth. These findings are relevant for a better understanding of the susceptibility of memory T cells to tolerizing environments. Situations of systemic and persistent Ag encounter may occur during persistent infections with viruses like HIV and hepatitis B or in individuals with widespread tumor burdens or malignancies of hemopoietic origin. Better understanding of the circumstances under which memory T cells lose their effectiveness to control pathogens or tumors is important for the development of safe intervention strategies to prevent or treat infectious diseases, cancer, or autoimmunity.


   Acknowledgments
 
We thank Michel Mulders for biotechnical assistance.


   Footnotes
 
1 This work was supported by Dutch Cancer Society Grants RUL 97-1450 and RUL 99-2025 and by a Royal Academy of Arts and Sciences fellowship (to R.E.M.T.). Back

2 Address correspondence and reprint requests to Dr. René E. M. Toes, Department of Rheumatology, Leiden University Medical Center, C4-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: r.e.m.toes{at}lumc.nl Back

3 Abbreviations used in this paper: tg, transgenic; LCMV, lymphocytic choriomeningitis virus; Ad5, adenovirus type 5; E1A, early region 1A. Back

Received for publication December 1, 2003. Accepted for publication March 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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