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The Journal of Immunology, 1999, 163: 330-336.
Copyright © 1999 by The American Association of Immunologists

Protective Immunity from Naive CD8+ T Cells Activated In Vitro with MHC Class I Binding Immunogenic Peptides and IL-2 in the Absence of Specialized APCs1

Conrad Hauser1,*,{dagger}, Frank Zipprich3,*, Isabelle Leblond*, Susanne Wirth* and Ambros W. Hügin{dagger}

* Allergy Unit, Division of Immunology and Allergy, and {dagger} Department of Dermatology, Hôpital Cantonal Universitaire, Geneva, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag-specific CTL can protect against tumors and some viral infections and may be useful for adoptive immunotherapy. Here, we show that purified CD8+ T cells from naive C57BL/6 mice can be primed in vitro with different immunogenic peptides, which bind to MHC class I gene products, and IL-2 to exhibit specific and MHC-restricted effector function in vitro and in vivo protection against lymphocytic choriomeningitis virus infection and B16.F10 melanoma lung metastases. Limiting dilution assays in the absence of feeder cells with highly purified CD8+ T cells from two transgenic mice strains, each expressing a different MHC class I-restricted TCR, indicated that only peptide and IL-2, but not TCR- cells, were required for the growth of naive CD8+ T cells. These alternative minimal requirements for the activation and expansion of specific CD8+ T lymphocytes, without the need for professional APC, may be exploited for adoptive immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The in vitro generation of specific responses to natural Ag in CD8+ T cells from naive animals has initially been regarded as impossible or very inefficient (1, 2). Exceptions are allo- or superantigen-driven responses that are thought to be due to high precursor frequency of allo- or superantigen-reactive cells within the naive T cell repertoire.

It is generally agreed that naive CD8+ T cells require costimulation from APC for induction of lymphokine production and CTL activity. Nussenzweig et al. (3) reported that dendritic cells could induce trinitrophenyl (TNP)-specific CTL function in T cells from nonprimed animals. Sprent and Schaefer (4) identified an APC for CD8+ T cells that remained poorly characterized. Cell-associated molecules as well as soluble mediators such as lymphokines may be responsible for this apparent dependence on APC. With the advent of molecular identification of costimulatory molecules and their receptors on T cells, it has been shown that several molecules can serve this function. There is good evidence that B7.1 (CD80) can function as costimulator for naive CD8+ T cells (5, 6, 7, 8, 9, 10, 11). 4-1BB (CD137) is a receptor expressed on T cells for an alternative costimulator molecule with a ligand (4-1BBL) expressed on certain APC. Signaling through 4-1BB preferentially coactivates CD8+ T cells (12). This costimulator pathway is CD28 independent but dependent on TNF receptor-associated factor 2 (13, 14). In contrast to B7.1, the heat stable Ag, another costimulator molecule expressed on some APC, was not required for induction of CTL from naive CD8+ T cells in vivo (15).

Recently, several groups reported that naive CD8+ T cells can be activated with immobilized MHC class I molecules and antigenic peptide (16, 17, 18, 19). However, in these studies the precursor frequency for specific T cells was high. Contamination by non-T cells in the responder T cell population and/or selective proliferation by CD8+CD44high (activated/memory) T cells was unlikely but not completely excluded.

We decided to study activation of naive CD8+ T cells by using MHC class I-restricted immunogenic peptides. Such peptides, derived from natural Ags, have been shown to represent TCR ligands when bound to MHC class I gene products. The purpose of this report is to demonstrate that that naive specific CD8+ T cells with a low precursor frequency, as it is the case in a natural naive T cell repertoire, can be activated with immunogenic peptide if IL-2 is provided. The second purpose is to demonstrate formally that non-T cells are not required for the activation of naive CD8+ T cells in vitro. To demonstrate this, we used limiting dilution experiments with naive T cells from mice with a transgenic (tg)4 TCR restricted to MHC class I gene products. It is unknown whether activation of naive CD8+ T cells without APC can generate biologically relevant T cells. Some authors have shown that activation and expansion of CD8+ T cells with high amounts of peptide selects for low-affinity T cells without protective function in vivo whereas only low amounts of peptide selected for high-affinity cells that can confer protection in vivo (20). Because we needed high concentrations of peptide to activate specific CD8+ T cells in vitro, the third purpose of this report is to demonstrate that protective effector cells can be generated in this way. To demonstrate this, we used a viral infection and a tumor model together with adoptive transfer of in vitro-activated CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides

The following peptides were synthesized and purified to >75% by HPLC. KAVYNFATM (lymphocytic choriomeningitis virus (LCMV) peptide) is derived from the sequence of the LCMV glycoprotein 33–41 (restriction elements Kb, Dd) except that C was replaced by M at position 41 (21, 22, 23). RGYVYQGL (vesicular stomatitis virus (VSV) peptide, restriction element Kb) is derived from the VSV nucleocapside 52–59 (24). VYDFFVWL (tyrosinase related protein-2 (TRP-2) peptide, restriction element Kb) was derived from the TRP-2 181–188 (25). YAMIYRNL (melanoma-derived peptide (mdm), restriction element Kb) was derived from the mdm-2 protein 100–107 (26). GGYKFGGL with a TNP substitution at the {epsilon}-amino group of K (TNP-peptide, restriction element Kb) was defined by Martin et al. (27). ASNENMDAM (influenza nucleoprotein (INP) peptide, Db) was derived from the INP 366–374 (28).

Mice

Mice were kept according to institutional guidelines and the experiments were done with permission of the state’s veterinarian office according to its guidelines. C57BL/6 mice were purchased from IFFA-Credo (L’Arbresle, France) or from BRL (Füllinsdorf, Switzerland). C57BL/6 mice with a tg TCR (P14) recognizing peptide 33–41 of the LCMV glycoprotein in the context of Db and Kd (21, 22, 23) were kindly provided by Drs. R. M. Zinkernagel and H. Hengartner (Institute of Experimental Immunology, Zürich, Switzerland). C57BL/6 mice with a tg TCR (F5) recognizing the peptide 366–374 of the INP in the context of Db (28) were kindly provided by Dr. J. Marvel (Ecole Normale Supérieur, Lyon, France) with the permission of Dr. D. Kioussis (National Institute for Medical Research, London, U.K.).

Cell preparation and culture

Lymph node cells from naive C57BL/6 mice were nylon wool filtered and subjected to negative selection with mAb to CD4 (GK1.5; American Type Culture Collection, Manassas, VA) and to MHC class II (M5/114.15.2; American Type Culture Collection) and complement. Alternatively, magnetic cell separation (Miltenyi, Bergisch Gladbach, Germany) with these mAb was performed. In some experiments, CD44 (polymorphic glycoprotein-1 (29)) was additionally used for negative selection. The resulting cells were 92–98% CD8+ (53-6.7; PharMingen, Palo Alto, CA). CD44high expressing cells were <1% when the mAb to CD44 was used and 7–11% CD44high when it was omitted. No CD11c+ (HL3, PharMingen), 33D1+ (30), or NLDC145+ (31) cells could be detected. The cells (2.5 x 105/well) were cultured at 37°C in 200 µl of DMEM (or IMDM for some of the limiting dilution and bulk culture experiments) containing 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin (all Life Technologies, Basel, Switzerland), 2 x 10-5 M 2-ME (Sigma, St. Louis, MO), 50 U/ml recombinant human (rh) IL-2 (Eurocetus, Amsterdam, The Netherlands), and 10-6 M different peptides in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark). After 5 days, pooled and washed cells were incubated in culture medium (1.5 ml/well) containing rhIL-2 (50 U/ml) at 1.5–2.0 x 105 cells/ml in 24-well culture plates (Nunc) for an additional 2–4 days.

In vitro effector cell assays

Primed and washed effector cells (5 x 104) were incubated with 105 RMA cells irradiated with 5000 rad in 200 µl of media in the presence or absence of the relevant or irrelevant peptide (all 10-6 M). IL-2 and IFN-{gamma} were determined in the 24 h supernatant as described (32, 33). One SD was <15% of the mean for both assays. IL-2 was measured with CTLL cells incubated with an 1:10 dilution of the supernatant and mAb to IL-4 (11B11 (34); kindly provided by Dr. W. E. Paul, National Institutes of Health, Bethesda, MD) to ensure that only IL-2 but not IL-4 was measured. The results are expressed as cpm proliferation of CTLL cells after 24 h incubation and addition of [methyl-3H]TdR for the final 4 h. For IFN-{gamma} detection in the supernatant of restimulated T cells, an ELISA using two mAb to IFN-{gamma} (35, 36), one for catching, the other biotinylated for detection by streptavidin coupled to HRP, was used. The sensitivity of the assay was 1 U/ml.

Washed effector T cells were incubated with 51Cr-labeled RMA cells (5 x 103/well) at the indicated E:T ratio in the presence or absence of 10-6 M of the indicated peptide. Release of radioactivity in the supernatant was determined after 4 h. Spontaneous release was usually <10% of the total release. Cytotoxicity was calculated as follows: (experimental release - spontaneous release) x 100%/(total release - spontaneous release). To some CTL assays, mAb to Kb (AF6–88.5; PharMingen) and/or to Db (H141.3 (37); kindly provided by Dr. H.R. MacDonald, Ludwid Institute for Cancer Research, Lausanne, Switzerland) was added. RMA cells (H-2b (38)) were provided by Dr. J. Maryanski, Ludwig Institute for Cancer Research) and cultured in DMEM containing 10% heat-inactivated FCS,100 U/ml penicillin, and 100 µg/ml streptomycin (all Life Technologies). The cells were subcultured three times per week and also the day before the CTL assay.

Limiting dilution assays

CD44high-depleted CD8+ T cells from either the P14 tg mice or the F5 tg mice were seeded at various cell numbers per well (24 wells/cell dilution) into round-bottom 96-well culture plates in media containing rhIL-2 (50 U/ml) and 10-6 M of the relevant or no peptide. After 1 wk (9 days in one experiment), the number of growing cell colonies was determined microscopically. In the case of the P14 tg cells, the plated cells were >=98% Vß8+ (MR5-2; PharMingen), the ß-chain family of the tg P14 TCR. In the case of the F5 tg mice, the plated cells were 99% Vß11+ (RR3-15; PharMingen), the ß-chain of the tg F5 TCR. No feeder cells were added. The media were the same as for bulk cultures.

In vivo protection assays

Four- to 8-wk-old C57BL/6 mice were injected i.v. with LCMV (strain WE, 200 pfu/animal). One day later, primed cells were administered i.v. to infected mice. The LCMV titer in the spleen was determined 4 days later as described (39). The number of injected effector cells varied between 3 and 6 x 106 per animal, depending on the experiment. Four- to 6-wk-old C57BL/6 mice were injected i.v. with 105 B16.F10 melanoma cells as described (25, 40). Two days later, in vitro primed effector cells (5–9 x 106 per animal) were injected i.v., and 16–18 days later, the lungs were removed and the macroscopically visible metastases were counted in a blinded fashion.

Statistics

Values of p of the log values from the LCMV foci (treated vs nontreated) were calculated with the two-way ANOVA. The mean of five independent experiments (three to five mice per group) are shown. The p values of the number of lung metastases (treated vs nontreated) were calculated with the two-way ANOVA modified by Brown-Forsythe. The mean count of lung metastases and SEM of five independent experiments (1 x 2, 3 x 3, and 1 x 4 mice per group) with injection of 5–9 x 106 cells per animal, depending on the experiment, is shown.

According to the Poisson model reported by Taswell (41), the probability of obtaining a positive result due to contamination by a TCR- cell in a single experiment is p = 1 - e-q, whereby q is the Poisson mean frequency of TCR- cells. Thus, the probability of obtaining a positive result in at least Z of N samples assayed in a single experiment can be computed according to the following binominal formula:

where k is the number of samples containing at least one TCR- cell.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specific effector responses can be induced in bulk cultures of naive CD8+ T cells by MHC class I binding peptides and IL-2

Purified CD8+ T cells from naive C57BL/6 mice were cultured with high concentrations (10-6 M) of MHC class I binding peptides, previously shown to be immunogenic (21, 22, 23, 24, 25, 26, 27), and rhIL-2. After 7–9 days of culture, >=97% of the cells recovered expressed the TCR-{alpha}ß, were CD44high, and expressed CD8. No NK1.1+ were detected and <=0.3% expressed the {gamma}{delta} TCR. Viable cells without measurable effector function were recovered from cultures with IL-2 but without exogenous peptide. Upon restimulation with RMA cells and peptide, release of IL-2 and IFN-{gamma} was inducible with the peptide used for in vitro priming (Fig. 1GoA) but not irrelevant peptides (not shown). CTL activities were also specific (Fig. 1GoB, see also Fig. 3GoC) and could be inhibited with mAb to the relevant MHC class I molecule in the CTL assay (Fig. 1GoC). No viable cells could be recovered from cultures without IL-2 even when the peptide was incubated. The addition of a mAb to the relevant MHC class I molecule at the time of priming prevented the induction of specific effector cells (not shown), and the incubation of mAb to CD8 at culture start prevented the recovery of viable cells. When fresh CD8+ T cells were further depleted of CD44high (memory/activated) cells, priming with peptide and IL-2 also induced Ag-responsive effector T cells (Fig. 1GoD). When peptide was titered in the priming culture, measurable specific CTL activity could be induced between 10-6 and 10-8 M of peptide (Fig. 2Go). The peptide designed as MHC class I anchor without covalently bound TNP hapten group did not elicit specific T cell responses when used for priming, even when dendritic cells were used for priming (not shown).



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  		FIGURE 1. Induction of Ag-dependent and MHC class I-restricted effector cells in vitro from CD8+ T cells of naive mice. CD8+ T cells purified from C57BL/6 mice were primed in vitro with peptide and IL-2. A, IL-2 (CTLL cell proliferation in supernatant diluted 1:5) and IFN-{gamma} were determined in the 24-h supernatant after restimulation with the relevant (filled bars) peptide or no peptide (open bars) and RMA cells. B–D, Standard CTL activity was determined (each panel shows a single representative from >=3 experiments). B, Lysis of RMA target cells by TNP-peptide primed cells in the presence of the TNP-peptide (TNP-pept), the same peptide without the TNP group (pept), or no peptide was performed. C, Cells were primed with the H-2Kb-restricted VSV peptide. The CTL assay was performed in the presence or absence of mAb to H-2Db ({alpha}-H-2Db, 10 µg/ml, H141-30) or to H-2Kb ({alpha}-H-2Kb, 10 µg/ml, BB-24) and in the presence or absence of VSV peptide (VSV). D, Purified CD8+ T cells (Total CD8) and cells additionally depleted of CD44high-expressing cells (CD44-depl) were primed with LCMV peptide. CTL activity was compared in the presence or absence of LCMV peptide (LCMV).

 


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  		FIGURE 3. In vitro activated and expanded specific effector T cells mediate protection against LCMV and B16.F10 melanoma. CD8+ T cells from naive C57BL/6 were primed in vitro using either the LCMV peptide (A and B), the TNP peptide (B), or a mixture of TRP-2 and mdm-2 peptides (C and D). LCMV peptide-primed cells (LCMV-T cells) (A and B) or TNP-peptide-primed cells (TNP-T cells) (B) were administered i.v. to mice infected with LCMV 1 day previously. The number of LCMV foci in the spleen was determined 4 days later. A, SEM was 495 LCMV foci for the PBS group and 129 LCMV foci for the LCMV group. C and D, Cells were primed with a mixture of the TRP-2 and mdm-2 melanoma peptides. C, CTL activity was determined in the presence of TRP-2 peptide (TRP-2) or mdm-2 peptide (mdm-2). D, Alternatively, melanoma peptide primed cells (T cells) were administered to mice injected i.v. with B16. F10 melanoma cells 2–3 days previously. The mean count of lung metastases and SEM is shown.

 


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  		FIGURE 2. CD8+ T cells from naive C57BL/6 mice were primed in vitro at the indicated concentrations of LCMV peptide. Five days later, the cells were washed and CTL assays were performed using RMA target cells and 10-6 M LCMV peptide (•) or no peptide ({circ}). The percent lysis at an E:T ratio of 20:1 is shown.

 
Naive CD8+ T cells from mice with tg MHC class I-restricted TCR grow in limiting dilution in the absence of contaminating non-T cells

Activation and proliferation of specific T cells with peptide and IL-2 in bulk cultures appeared to occur in the absence of specialized APC as only purified CD8+ T cells were used. But contamination of the bulk T cell preparations by small numbers of non-T cells which may act as APC could not be ruled out. For this reason and to positively demonstrate that non-T cells were not required for activation and growth of CD8+ T cells, we used limiting dilution assays. To have a precursor frequency of specific CD8+ T cells that is higher than that of non-T cells in the starting cell population, we chose TCR tg mice bred into the C57BL/6 background. CD44lowCD8+ T cells were prepared from P14 and F5 TCR tg mice (22, 23, 28) and cultured with or without the relevant peptide. The fraction of wells with microscopically detectable colonies was determined (Table IGo). The probability of colony growth due to contamination by a non-T cell was calculated at low cell density input (<=14 cells/well), taking the purity of the starting T cell population into account (1–2%). The result of these calculations (Table IGo) indicated that tg CD8+ T cells were activated and grew to form colonies in the presence of peptide plus IL-2 and in the absence of contaminating non-T cells. Because the relative number of contaminating CD44high cells was lower than of non-T cells (<1%), the probability that colony formation at <=14 cells/well was exclusively due to growth of CD44high cells was even lower than the probability of non-T cell contamination (calculations not shown).


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  		Table I. Colony count of CD44low tg CD8+ T cells cultured in limiting dilution1

 
In vitro primed CD8+ T cells confer immunological protection in vivo

We next asked whether in vitro primed T cells could confer protective immunity in vivo. To this end, in vitro primed CD8+ effectors were tested in a model of infection by LCMV, a noncytopathic virus mainly controlled by CD8+ T cells and perforin (42, 43), and in a model of tumor inoculation with B16.F10 melanoma cells (25, 40). In vitro-primed cells were injected into naive mice infected previously with LCMV. The viral titer in the spleen, determined 4 days later, was significantly decreased compared with animals receiving no T cells (p < 0.0001; Fig. 3GoA). T cells primed to an irrelevant peptide did not protect (Fig. 3GoB). The i.v. transfer of CD8+ T cells primed in vitro with a mixture of the TRP-2 and the mdm-2 melanoma peptides into mice previously inoculated with B16.F10 melanoma cells significantly decreased the number of lung metastases determined 16–18 days later (range, 27–88% reduction over control, p < 0.025; Fig. 3GoD). Together, these results demonstrate that T cells primed in vitro for a single round with peptide and IL-2 have a protective effect in vivo.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both peptide and IL-2 were required for successful in vitro priming. Peptide had to be added at high concentrations and thus probably provided sufficient TCR ligand density for activation of CD8+ T cells in the presence of IL-2. These conditions were apparently also sufficient to bypass the requirement for accessory signals. The addition of IL-2 to CD8+ T cells has previously been reported by Harding and Allison to replace the requirement for CD28 ligation in the priming for allospecifc CTL responses (5). Different results were reported by Guerder et al. (6) who found that the blocking of B7 by CTLA4-Ig fusion protein resulted in diminished but not absent induction of CTL function in CD44lowCD8+ T cells. The addition of IL-2 abrogated to some extent the blocking by CTLA4-Ig fusion protein in CD44highCD8+ but not in CD44lowCD8+ T cells. Guerder et al. used APC from a tg Ag-expressing mouse line and syngeneic TCR tg CD8+ T cells. The results of Harding and Allison do not rule out induction of CTL from CD44highCD8+ T cells because they did not separate their CD8 T cell preparation into CD44high and CD44low fractions.

Activation and proliferation of naive CD8+ T cells recognizing a MHC class I binding peptide in the apparent absence of APC has previously been postulated based on a number of arguments (16, 17, 18, 19). In three of the reports (16, 17, 18), the authors used a tg MHC class I-restricted TCR that was bred into an unrelated MHC. This rendered impossible the generation of signal 1 but not of signal 2 by contaminating APC. All groups observed T cell proliferation in bulk culture using immobilized MHC class I plus peptide. It was thus possible that contaminating APC in the responder population provided signal 2 in these cultures. Another possibility is that contaminating CD44high cells proliferated in bulk cultures, given the high cell inputs and the relatively low levels of thymidine incorporation reported by these authors. It is of note in this context that they used responder T cells with high or very high (close to 1) precursor frequency in bulk culture. In only one report (16) T cell proliferation T cells was determined at low cell numbers (>=1500 cells/well). In these experiments, the T cells were >98.5% CD8+. A contamination by non-T cell APC of an average of 1% would still represent an average contamination by non-T cells of 15 cells/well when 1500 cells are seeded. In addition, it is unknown whether the CD8+ cell fraction contained lymphoid dendritic cells that can express CD8. Calculations from limiting dilution experiments performed by the same group at 0.3, 3, and 3 cells/well yielded a plating efficiency of 1:13. These results agree with ours. Unfortunately, the limiting dilution experiments by Pardigon et al. could leave some doubt on the validity of the conclusions because the percentage of CD44high cells used for these experiments was not reported. In an earlier section of this report, it was mentioned that >85% of the T cells were CD44low, leaving the possibility of an up to 15% CD44high cell fraction. We believe that the direct calculation of the probability of non-T cell contamination and the probability of exclusive growth of CD44high T cells at low seeding cell density (1–10 cells/well) as shown here is robust. Together, our conclusions agree with that of other groups in that CD8+CD44low cells mice with MHC class I-restricted tg TCR can be activated without APC.

Another difference between our culture system and that of the authors cited above (16, 17, 18, 19) is the requirement for IL-2 in our system as opposed to proliferation of CD8+ T cells in response to immobilized MHC class I plus peptide without addition of exogenous IL-2. One explanation is that, compared with our system, engineered and immobilized MHC class I containing immunogenic peptide may provide a much higher TCR ligand density, a stronger signal 1, and thus independence from exogenous IL-2. Exogenous peptide when added to CD8+ T cells needs to compete with endogenously loaded peptide to generate a TCR ligand. The lower TCR ligand density generated in our system may explain why IL-2 was required.

T cells express MHC class I molecules and most likely functioned here as presenting cells. It remains to be determined whether T cells presented peptide to each other or if a single T cell can present peptide to itself. Nonetheless, there are in vivo situations in which nonprofessional APC appear to induce protective CD8 and perforin-dependent immunity. Fibroblasts presenting the LCMV peptide can induce protection if injected into lymphoid organs, but not if injected into the foot pad (21). Using the same model, it has been demonstrated that CD28-deficient mice can mount a protective immune response to LCMV under certain priming conditions (44).

Using MHC class I peptide constructs, it has recently been shown that precursor frequency determinations by limiting dilution analysis underestimated the abundance of specific T cells (45). This is compatible with our observation that cell culture inputs used here can regularly yield specific effector T cells. In addition to the demonstration of specific effector cell function in vitro, we answered the crucial question of whether T cells activated with high concentrations of peptide and IL-2 are capable of mediating protective immunity. In both models, we observed protection. However, the protection was not complete. It remains to be determined whether optimization of culture conditions for the expansion of specific T cells can result in complete protection.

Activation and expansion of specific CD8+ T cells without the need for professional APC also has implications for immunotherapy in humans. It may provide a valuable alternative to vaccination with Ag bearing dendritic cells. An obvious advantage of this method compared with vaccination with dendritic cells is that the induction of effector function can be controlled in vitro. Furthermore, activation of T cells with APC expressing the costimulators B7-1 or B7-2 such as dendritic cells may inhibit the proliferation of specific CTL precursors with high-affinity/avidity (9).


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  		Table 1B.

 

   Acknowledgments
 
We thank Drs. H. Hengartner, J. Marvel, and D. Kioussis for tg mice, Drs. H. R. MacDonald, H. Hengartner, and J. Marvel for reagents, Dr. H. R. MacDonald for critical reading of the manuscript, and Drs. S. Bucher and B. Mermillod for help with the statistics.


   Footnotes
 
1 This work was supported by a grant from the Swiss National Foundation for Scientific Research (Grant 31–40466.94 to C.H.), the Schweizerische Krebshilfe (to A.W.H and C.H.), and the Deutsche Forschungsgemeinschaft (to F.Z.). Back

2 Address correspondence and reprint requests to Dr. Conrad Hauser, Allergy Unit, Hôpital Cantonal Universitaire, 1211 Geneva 14, Switzerland. E-mail address: Back

3 Current address: Department of Dermatology, University of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. Back

4 Abbreviations used in this paper: tg, transgenic; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus; TRP, tryosinase-related protein; mdm, melanoma-derived peptide; TNP, trinitrophenyl; INP, influenza nucleoprotein; rh, recombinant human. Back

Received for publication December 17, 1998. Accepted for publication April 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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