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The Journal of Immunology, 2002, 169: 3053-3060.
Copyright © 2002 by The American Association of Immunologists

TNF-Mediated Toxicity After Massive Induction of Specific CD8+ T Cells Following Immunization of Mice with a Tumor-Specific Peptide1

Janine Bilsborough2,3,*, Catherine Uyttenhove2,*, Didier Colau*, Philippe Bousso{ddagger}, Claude Libert§, Birgit Weynand{dagger}, Thierry Boon* and Benoit J. Van den Eynde4,*

* Ludwig Institute for Cancer Research and Cellular Genetics Unit and {dagger} Department of Pathology, Université de Louvain, Brussels, Belgium; {ddagger} Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale, Unité 277, Paris, France; and § Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We immunized mice with antigenic peptide P815E, which is presented by H-2Kd and recognized by tumor-specific CTL raised against P815 tumor cells. This peptide is encoded by the ubiquitously expressed gene MsrA and carries a mutated residue conferring tumor specificity. Unexpectedly, we observed a severe toxicity occurring in the early hours after the third injection, resulting in the death of most mice within 24 h. The toxic syndrome was reminiscent of TNF-induced shock, and the sera of ill mice contained high levels of TNF. Toxicity was prevented by injection of neutralizing anti-TNF Abs, confirming the involvement of TNF. Depletion of CD8+ T cells could also prevent toxicity, and ex vivo experiments confirmed that CD8+ lymphocytes were the major cellular source of TNF in immunized mice. Tetramer analysis of the lymphocytes of immunized mice indicated a massive expansion of P815E-specific T cells, up to >60% of circulating CD8+ lymphocytes. A similar toxicity was observed after massive expansion of specific CD8+ T cells following immunization with another P815 peptide, which is encoded by gene P1A and was injected in a form covalently linked to an immunostimulatory peptide derived from IL-1. We conclude that the toxicity is caused by specific CD8+ lymphocytes, which are extensively amplified by peptide immunization in a QS21-based adjuvant and produce toxic levels of TNF upon further stimulation with the peptide. Our results suggest that immunotherapy trials involving new peptides should be pursued with caution and should include a careful monitoring of the T cell response.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To better understand the processes required to induce antitumor immunity, we have used a murine mastocytoma tumor model that has been widely used for the study of antitumor immune responses. Murine mastocytoma P815 can induce CTL responses to at least four distinct Ags (AB, C, D, and E). P815AB was the first of these Ags to be identified. It belongs to the category of tumor-specific common Ags and is encoded by gene P1A, a gene silent in normal tissues except in spermatogonia, which do not express MHC class I molecules (1, 2). P815E, the second Ag to be identified on P815, is a tumor-specific individual Ag that arises as a result of a mutation within an ubiquitously expressed gene known as methionine sulfoxide reductase (MsrA) (3).

In previous years, Ag P815AB has been the focus of a number of studies as a model for designing and optimizing antitumor vaccines, including vaccines based on naked DNA, synthetic peptide, purified protein, pulsed dendritic cells, and recombinant viruses (4, 5, 6, 7, 8, 9). CTL responses against Ag P815AB were achieved with naked DNA (6), viral vectors (10), or peptide-pulsed APC injected with IL-12 (8). The strongest CTL responses were obtained after injection of living cells of the MHC-matched leukemia line L1210, previously transfected with genes P1A and B7-1 (7). Such responses provided protection against a challenge with a lethal dose of P815 tumor cells, indicating that P815AB is a major tumor rejection Ag on P815 (6, 7, 8, 10). In contrast, protection against P815 challenge could not be achieved using L1210 leukemia cells transfected with P815E and B7-1 (L1210.P1E.B7-1), even though Ag-specific CTL were generated as a result of immunization (3). We have previously demonstrated that strong CTL responses against tumor-specific Ags could also be induced with synthetic peptides combined with IL-12 and an adjuvant containing QS21 and monophosphoryl lipid A (MPL)5 (9). Used with the P1A peptide, this protocol was well tolerated and induced strong CTL responses against Ag P815AB. We therefore used this protocol to immunize mice with peptide P815E for the purpose of assessing the induction of P815E-specific CTL in vivo and evaluating protection against tumor challenge. We report here that considerable toxicity was observed.


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

Syngeneic DBA/2 mice were raised in specific pathogen-free conditions. All mice used in these experiments were between 8 and 12 wk old.

Peptides

All peptides were synthesized in-house on solid phase using F-moc for transient NH2-terminal protection according to the study of Atherton et al. (11) and were characterized using mass spectrometry. All peptides used in this study were purified by HPLC to >99% homogeneity. They were stored at -20°C at 20 mg/ml in DMSO. The single-letter code sequences of the peptides used are as follows: H-2Kd-restricted P815E, GYCGLRGTGV (3); H-2Ld-restricted P815A (positions 35–43 of the P1A protein), LPYLGWLVF (12); hybrid peptide P1A-VIL1, LPYLGWLVFVQGEESNDK (13); and H-2Kd-restricted P198, KYQAVTTTL (14).

Cell lines

All of the experiments using cells referred to as P815 were performed with an azaguanine-resistant clone of cell line P815, named P511. Cell line L1210.P1E.B7-1 has been described previously (3). All cell lines were cultured in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS in a humidified, 8% CO2 atmosphere at 37°C.

Adjuvant

The SBAS-1c adjuvant, comprising MPL and liposomal DQS21 (Quilla saponaria, fraction 21) was kindly prepared by Dr. S. Cayphas (Preclinical Operations-Formulation; Glaxo-SmithKline Biologicals, Rixensart, Belgium). MPL is a detoxified version of lipid A extracted from Salmonella minnesota. DQS21 stands for detoxified QS21 where the lytic activity of QS21 is quenched by the addition of lipids. An adjuvant containing MPL and QS21 in an emulsion was used in humans in a clinical trial of malaria vaccine described in the report by Stoute et al. (15).

Recombinant murine (m)IL-12

Murine rIL-12 (His)6 was partially purified from the supernatant of P1.HTR cells transfected with the p40 and p35 (His)6 cDNA plasmids (16, 17) as described elsewhere (9). The biological activity of recombinant mIL-12 was measured as previously described (18). One unit per milliliter was defined as the concentration that stimulated half-maximal proliferation of C57BL/6 murine Con A blasts. The specific activity of IL-12 estimated using this test was between 10 and 15 x 106 U/mg for batches obtained after affinity chromatography.

Immunization of mice

Unless otherwise stated, mice were immunized s.c. in the footpads with 50 µg of peptide mixed with adjuvant SBAS-1c containing 300 U of IL-12 on day 0. This immunization was followed on days 1 and 2 by injections of 300 U of IL-12 in the footpads. For the experiments using peptides P1A and P1A-VIL1, an equimolar dose of both peptides, i.e., 25 µg of peptide P1A and 50 µg of peptide P1A-VIL1, was injected with adjuvant SBAS-1c in the absence of any IL-12 injection.

In vivo depletion of T cell subsets

Mice were injected i.p. with 1 ml of cell-free ascitic fluid of GK1.5 (anti-CD4) or 53.6.72 (anti-CD8), 7, 6, and 5 days before and 5 days after the first peptide immunization. Splenocytes were collected from animals 1 wk following this treatment and tested for the presence of CD4+ or CD8+ subsets by FACS analysis. Cell subsets were depleted to >95%.

Isolation of cells by MACS

Splenocytes were collected from mice that had been immunized with peptide plus adjuvant and IL-12 1 h previously. Cells were resuspended in degassed buffer (PBS supplemented with 0.5% BSA) at a concentration of 107 cells/90 µl. Anti-CD8 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to cells at 10 µl/107 total cells and incubated for 15 min at 4°C. After incubation the cells were washed and CD8+ cells were isolated using magnetic separation columns (Miltenyi Biotec). The CD8- fraction was collected and resuspended at 107 cells/90 µl of buffer. Anti-CD4 magnetic beads were added at 10 µl of beads/107 total cells, and CD4+ cells were isolated. Anti-B220 magnetic beads were added to the remaining cells and B220+ lymphocytes were isolated. The isolated cells were put into culture at 4 x 105 cells/well. Supernatant was collected from all wells 2 h later. This supernatant was tested for the presence of TNF using WEHI-164 clone13 cells as described below.

Production of tetramers H-2Kd-P815E and H-2Ld-P815A

Plasmids for expression in Escherichia coli of the extracellular H-2Kd and H-2Ld domains having a birA biotinylation site at their carboxyl-terminal ends were prepared as previously described (19). H-2Kd and H-2Ld complexes were folded in vitro with {beta}2-microglobulin and, respectively, peptide P1E (GYCGLRGTGV) and P1A (LPYLGWLVF) as described previously (20). Soluble complexes, purified by gel filtration, were biotinylated using the birA enzyme (Avidity, Denver, CO). PE-labeled tetramers were produced by mixing the biotinylated complexes with extravidin-PE (Sigma-Aldrich, St. Louis, MO).

Tetramer staining and FACS analysis

Cells were stained (106 cells/ml) for 15 min at room temperature in FACS buffer containing 1% FCS, 5 µg/ml anti-mouse CD16/CD32 (BD PharMingen, San Diego, CA) and the appropriate PE-tetramer at 50–100 nM H-2 molecules. Then anti-CD3-FITC (BD PharMingen) and anti-CD8-PerCP (BD PharMingen) labeled Abs were added and cells were incubated for 15 additional min at room temperature. Cells were washed in FACS buffer and analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA).

Cloning of H-2Kd-P815E-tetramer-positive cells

PBL were isolated from mice 15 days after immunization with 106 living L1210.P1E.B7-1 cells i.p. or 10 days after the second injection of peptide P815E with adjuvant and IL-12. Single CD8+CD3+ H-2Kd-P815E-tetramer+ cells were isolated by FACS and seeded at 1 cell/well in 96-well round-bottom plates (Nunc, Roskilde, Denmark) containing 50 µl of medium. Cells were then stimulated weekly with a mix containing 5 x 105 irradiated splenocytes and 3 x 103 irradiated L1210.P1E.B7-1 cells pulsed or not with peptide in medium containing 50% secondary MLC supernatant and supplemented with 20 U/ml recombinant human IL-2. CTL clones appeared 2–3 wk after the first stimulation.

Collection of plasma for kinetic studies

Mice were heated in an incubator at 42°C for 30 min before collection of blood samples. A small sample of blood was collected via the tail vein in a 75-mm/75-µl hematocrit-capillary tube containing sodium-heparin (Hirschmann Laboratories, Eberstadt, Germany). The hematocrit tube was centrifuged using a capillary tube rotor at 8000 rpm for 5 min. Separated plasma was collected into small Eppendorf tubes and stored at -20°C before testing. In the experiment reported in Fig. 6Go, mice were bled at the retro-orbital sinus. Blood samples were incubated 30 min at 37°C to allow clotting before centrifugation at 10,000 rpm for 5 min. Sera samples were stored at -20°C in flat-bottom microplates before testing.



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  		FIGURE 6. TNF levels in the sera of mice immunized with chimeric peptide P1A-VIL1. Mice received several immunizations with the indicated peptide in adjuvant in the absence of IL-12. Of the 15 mice that received chimeric peptide P1A-VIL1, 3 died after four injections and 6 after five injections. Serum samples were collected in three surviving animals before and at four time points after a sixth injection of chimeric peptide. Four mice receiving six injections of the P1A peptide were tested similarly. TNF levels were estimated using TNF-sensitive WEHI-164 cells in a MTT colorimetric assay as described in Materials and Methods. {dagger}, Death of the mouse.

 
Treatment with anti-TNF-{alpha} Abs

Inhibiting anti-mTNF-{alpha} serum was produced in rabbits as follows: two female New Zealand albino rabbits (Animalabo, Brussels, Belgium) were injected with 10 µg of recombinant mTNF (sp. act. of 1.6 x 108 IU/mg, expressed and purified from E. coli in our laboratories) in combination with FCA (Difco, Detroit, MI), s.c. in legs and back. Rabbits were bled and boosted with 10 µg of TNF 4 and 8 wk after the priming injection. Titers were maximal after the second boost: ~30,000 inhibitory U/ml serum. These units are defined as the dilution of the antiserum needed to inhibit 1 IU/ml mTNF. Fifty microliters of the antiserum completely protected mice from a lethal challenge of LPS in combination with the sensitizing D-(+)-galactosamine. The antiserum was injected i.p. 2 h before the third immunization with peptide P815E.

Thalidomide treatment

Mice were injected i.p with 100 mg/kg thalidomide (Sigma-Aldrich) dissolved in PBS.

TNF assay

Plasma samples from mice were serially diluted before addition to wells of flat-bottom 96-well plates. The level of TNF present in each plasma sample was assessed using the TNF-sensitive WEHI-164 clone 13 (21) in a MTT colorimetric assay (22). TNF quantification was made possible by the addition of a LT standard curve and all samples were tested at the same time.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunization of mice with peptide P815E induces a lethal toxicity

DBA/2 mice were injected s.c. in the footpad at weekly intervals with 50 µg of purified synthetic peptide P815E combined with a human-use adjuvant and 300 U of IL-12. Surprisingly, we observed that after the third immunization the majority of mice suffered from a toxic shock-type syndrome resulting in death within 24 h after the injection. The mice that survived the third immunization succumbed after a fourth injection (Fig. 1GoA). The first signs of peptide-induced toxicity usually appeared within 5 h of peptide immunization. The symptoms included loss of temperature (~4°C below normal), diarrhea, piloerection, lethargy, and loss of balance.



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  		FIGURE 1. Toxic shock after immunization with peptide P815E. DBA/2 mice were immunized following the indicated scheme where arrows indicate days of immunization. All mice were immunized twice with peptide P815E, adjuvant, and IL-12 before receiving additional injections of either peptide P815E with adjuvant and IL-12 (A), PBS with adjuvant and IL-12 (B), peptide P815E with adjuvant (C), peptide P815E with IL-12 (D), peptide P815E alone (E), wild-type peptide wtMsrA with adjuvant and IL-12 (F), or L1210.P1E. B7-1 cells (G). The number of dead mice is indicated. All of the dead mice died within 24 h after the peptide injection, usually even within the first 8 h. In group A, 13 of 18 mice died after the third injection and the remaining 5 mice died after the fourth injection. All of the mice that did not die within 24 h after the last injection recovered completely and survived until the end of the experiment. Peptide injections (50 µg/mouse) were performed s.c. in the footpads, whereas the injection of L1210. P1E. B7-1 cells was performed i.p. (106 cells/mouse).

 
This toxicity was not caused directly by the adjuvant/IL-12 combination, as mice receiving two complete immunizations (peptide/adjuvant/IL-12) followed by two injections of adjuvant/IL-12 without peptide did not show any signs of toxicity (Fig. 1GoB). Moreover a third injection with peptide P815E alone was sufficient to induce toxicity in mice having received two complete immunizations, further demonstrating that the toxicity was not caused directly by the adjuvant and/or IL-12 (Fig. 1Go, C–E). This, along with our previous observation that the same immunization mode used with other peptides does not cause such toxicity, indicated that the toxicity was related to the response to the P815E peptide (9).

We previously observed that the peptide corresponding to the nonmutated sequence of the MsrA gene (wtMsrA) was capable of binding to H-2Kd (3). It was possible therefore that the toxicity resulted from the induction of an immune response cross-reactive with the wild-type peptide, thereby resulting in autoimmunity. However, we observed no toxicity when mice received two complete immunizations with peptide P815E followed by multiple boost injections with the wild-type MsrA peptide combined with adjuvant and IL-12 (Fig. 1GoF).

Role of TNF in the peptide-induced toxicity

Histological examination of tissues taken from animals before death showed evidence of hepatic panlobular necrosis due to microthrombi formation, acute tubular necrosis, "ischemic" colitis, and enteritis. Moreover, a large number of circulating cells comprised mostly of blastic CD3+ cells with a small number of CD45R+ cells (presumably B cells) and F4/80+ cells (macrophages) were present in the blood vessels of peptide-immunized animals but not in samples from normal mice (data not shown). The symptoms listed above were similar to those reported for anti-CD3-mediated toxicity and LPS-induced shock, which are both known to induce a rapid increase in circulating TNF-{alpha} levels (23, 24).

We therefore tested TNF levels in the plasma of animals collected at four time points (30 min, 4 h, 8 h, and 24 h) following the last peptide immunization and compared these levels with those in plasma collected the day before immunization. Groups of animals were immunized with adjuvant plus IL-12 along with peptide P815E, PBS, or peptide P198, which also results from a mutation and is expressed by an immunogenic variant derived from P815 tumor cells (14). The latter peptide does not cause toxicity when injected in those conditions (9). The results showed a peak in TNF production 30 min after immunization for all animals, including those given PBS plus adjuvant and IL-12 (Fig. 2Go). TNF levels in animals immunized with PBS or P198 dropped quickly to 221 and 280 pg/ml, respectively, at the 4-h time point (means of 4 and 9 animals, respectively). However, TNF levels in plasma of P815E-peptide-immunized mice remained elevated, with a level of 9379 pg/ml at the 4-h time point (mean of eight animals) (Fig. 2Go). Sustained high levels of circulating TNF were also observed in mice receiving peptide without adjuvant and IL-12 at the third injection, with a level of 19,526 pg/ml at the 4-h time point (mean of six animals).



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  		FIGURE 2. TNF levels in the plasma of peptide-immunized mice. DBA/2 mice were injected with peptide P815E (three injections, n = 5; or four injections, n = 5), PBS (four injections, n = 4), or peptide P198 (five injections, n = 8), all mixed with adjuvant and IL-12. Another group of mice (n = 10) received two injections of peptide P815E with adjuvant and IL-12, followed by a third injection of peptide P815E alone. Plasma samples were collected via the tail vein for each animal 24 h before (-24) and at four time points (30 min, 4 h, 8 h, and 24 h) after the last injection (arrow). TNF levels were estimated using TNF-sensitive WEHI cells in a MTT colorimetric assay as described in Materials and Methods. {dagger}, Death of the mouse.

 
To determine whether those sustained levels of TNF were responsible for the lethal toxicity observed, we injected mice i.p. with neutralizing anti-TNF-{alpha} Abs 2 h before a third immunization with peptide P815E with adjuvant and IL-12. As shown in Table IGo, the highest dose of anti-TNF Abs provided complete protection against the lethal toxicity. This was also true with mice receiving peptide P815E alone at the third injection, in which 10 of 10 mice were protected with 100 µl of Abs as opposed to 9 of 11 dead mice in the control group (data not shown). The role of TNF in toxicity was further confirmed when we observed that treatment of mice with thalidomide, which is known to inhibit TNF-{alpha} production, allowed all mice to survive a third, a fourth, and even a fifth immunization with peptide P815E (Table IGo) (25, 26).


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  		Table I. Protective effect of anti-TNF Abs or thalidomidea

 
CD8+ lymphocytes are the major source of TNF

Soluble TNF has been shown to play a significant role in bacterial endotoxin- and exotoxin-induced toxic shock (27, 28, 29). Lymphocytes appear to be the major source of TNF in the shock induced by bacterial exotoxins such as staphylococcal enterotoxin B, which have superantigenic properties resulting in massive T cell proliferation. SCID mice are not sensitive to staphylococcal enterotoxin B and only become sensitive upon T cell repopulation (30). On the other hand, SCID mice are sensitive to the shock induced by endotoxins such as LPS, which appear to trigger TNF production by macrophages (30).

We observed that SCID mice were not sensitive to the toxicity induced by immunization with peptide P815E, indicating that lymphocytes were most likely required for lethality (data not shown). The toxic response was observed in various strains of immunocompetent H-2d animals, although BALB/c mice were less sensitive than B10.D2 or DBA/2 animals (data not shown).

To confirm the involvement of lymphocytes and to determine which T cell subset may be directly implicated in TNF production, we isolated CD8+ T cells, CD4+ T cells, and B lymphocytes (B220+) from the spleen of animals 1 h after the third immunization with peptide P815E. Cell subsets were collected by magnetic separation and incubated in culture medium for 2 h at 37°C. Comparison of the TNF levels in the supernatant showed that the CD8+ lymphocyte population secreted most of the TNF (Fig. 3GoA). In agreement with those results, we observed that mice depleted of CD8+ lymphocytes were completely protected against toxic shock, whereas CD4+-depleted mice were not (Fig. 3GoB). TNF levels in the plasma of CD8+-depleted animals were lower than in the plasma of intact mice or CD4+-depleted mice, which is in line with the notion that TNF is the main mediator of toxicity (Fig. 3GoB).



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  		FIGURE 3. Role of CD8+ lymphocytes in TNF production and P815E-peptide-induced toxicity. A, Lymphocyte cell subsets were isolated from splenocytes of animals immunized with either peptide P815E combined with adjuvant and IL-12 (n = 2) or PBS with adjuvant and IL-12 (n = 2). Lymphocyte subsets were isolated using magnetic beads and MACS high-gradient magnetic columns. Immediately following isolation, cells were placed in culture with medium at 37°C. Two hours later, the supernatant was collected and TNF was measured using TNF-sensitive WEHI cells in a MTT colorimetric assay. B, DBA/2 mice were depleted of either CD4+ (n = 4) or CD8+ (n = 4) cells or were left untreated (n = 4) and immunized three times at weekly intervals with peptide P815E combined with adjuvant and IL-12. Plasma samples were collected via the tail vein for each animal at 24 h before (-24) and four time points after the third immunization (30 min, 4 h, 8 h, and 24 h). TNF levels were estimated using TNF-sensitive WEHI cells in a MTT colorimetric assay. {dagger}, Death of the mouse. Depletion was performed by i.p. injections of Abs GK1.5 (anti-CD4) or 53.6-72 (anti-CD8) 7, 6, and 5 days before and 5 days after the first peptide immunization. Inset, The result of an identical immunization experiment where toxicity rather than the TNF level was monitored. The number of dead mice in each group after three immunizations is indicated.

 
Massive expansion of anti-P815E CD8+ T cells in peptide-immunized mice

The development of toxicity was dependent on the presence of the P815E peptide in the immunization mixture and CD8+ lymphocytes in the recipient mouse, which suggested the involvement of P815E-specific CD8+ lymphocytes. We used H-2Kd-P815E tetramers to estimate the number of P815E-specific CD8+ T cells among blood lymphocytes (PBL) after the second peptide immunization by ex vivo FACS analysis. Between 45 and 75% of CD8+ blood lymphocytes were labeled with the tetramer, indicating a massive expansion of Ag-specific CTL (Fig. 4Go). In contrast, the expansion of tetramer-positive cells in the PBL of mice immunized with L1210.P1E.B7-1 cells was much lower, which is in line with the lack of toxicity after immunization with those cells (Fig. 4Go) (3). These data therefore suggest that development of toxicity is determined by the absolute number of specific T cells induced by the immunization.



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  		FIGURE 4. Expansion of H-2Kd-P815E tetramer-positive CD8+ T cells after peptide immunization. DBA/2 mice were immunized either with peptide P815E or with cells expressing Ag P815E (L1210.P1E.B7-1). Blood lymphocytes were collected 7–12 days after the immunization and stained with H-2Kd-P815E tetramers, with anti-CD8 and anti-CD3 Abs. Each • represents the percentage of tetramer-positive cells among CD8+ lymphocytes of an individual mouse. All tetramer-positive cells were CD8+. The average percentage of CD8+ among CD3+ cells was 29.9 in peptide-immunized mice and 17.3 in mice immunized with cells. Peptide immunizations were performed twice 7 days apart with 50 µg of peptide P815E in adjuvant and IL-12, and blood lymphocytes were collected for the tetramer staining 7 days after the second immunization. Immunizations with L1210. P1E.B7-1 cells were performed by injecting 106 living cells i.p. 12 days before the analysis. In a separate experiment, we observed that a second immunization with L1210.P1E.B7-1 did not increase the number of tetramer-positive cells. Control mice received either no injection (10 mice) or two injections of PBS with adjuvant and IL-12 (4 mice).

 
To determine whether the massive expansion of P815E-specific CTL was the sole factor determining toxicity or whether the CTL induced after peptide immunization were also qualitatively different, we compared the activity of P815E-specific CTL clones isolated from mice immunized either with peptide P815E/adjuvant/IL-12 or with L1210.P1E.B7-1 cells. CD3+CD8+H-2Kd-P815E-positive CTL were isolated and cloned directly from the blood of immunized animals using FACS. A total of 36 growing clones were obtained, 25 from mice immunized with peptide and 11 from mice immunized with L1210.P1E.B7-1 cells. They were tested for lytic activity and TNF production against P815 cells pulsed or not with peptide P815E. All clones were equally able to lyse P815 cells (data not shown) and produce TNF upon stimulation by P815 cells (Fig. 5Go). Although it is not impossible that the in vitro amplification has modified the activity of the clones, these results suggest that there is no qualitative difference in the effector function of the CD8+ cells induced by the two types of immunization, and therefore the absolute number of CD8+ cells appears to be the sole factor responsible for toxicity after peptide immunization. Irrespective of their origin, all clones also produced much more TNF after stimulation with peptide-pulsed P815 cells than with unpulsed cells (Fig. 5Go). This could explain why no toxicity was observed when L1210.P1E.B7-1 cells were injected into mice immunized twice with peptide/adjuvant/IL-12 (Fig. 1GoG).



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  		FIGURE 5. Identical TNF secretion profile of P815E-specific CD8+ clones isolated from mice immunized with peptide P815E ({circ}) or with L1210.P1E.B7-1 cells (•). Mice were immunized either with peptide P815E or with L1210.P1E. B7-1 as indicated in the legend of Fig. 4Go. Blood lymphocytes were collected 10–15 days after the immunization, stained with H-2Kd-P815E tetramers, sorted by FACS, and seeded at 1 cell/microwell. Clones were amplified in vitro and tested for TNF production after stimulation in vitro with P815 cells pulsed or not with peptide P815E. TNF production was measured after overnight incubation using TNF-sensitive cells WEHI-164 clone 13.

 
Immunization with another tumor-derived peptide can also induce toxicity

To determine whether the toxicity we observed after massive expansion of specific CD8+ T cells was peculiar to Ag P815E or would also occur with other antigenic peptides, we used Ag P815A, the other defined Ag of tumor P815, which is encoded by gene P1A and presented by H-2Ld (12). A number of reports have described various immunization protocols against this Ag, some of them leading to antitumor effects, without detectable toxicity (5, 6, 7, 9). To induce a greater expansion of CD8+ T cells against Ag P815A, we immunized mice with a chimeric peptide (P1A-VIL1) composed of the P1A antigenic peptide of nine amino acids covalently linked at its C terminus to a nine-residue peptide derived from human IL-1 {beta}, which has been reported to have the potent immunostimulatory effects of IL-1 without its toxicity (13). We observed the sudden death of 9 of 10 mice after the fourth immunization with the chimeric peptide, whereas mice receiving the P1A antigenic peptide alone or the IL-1 peptide alone were normal. In repeated experiments, we observed varying proportions of mice dying after the fourth injection of P1A-VIL1 (3/10, 9/10, 9/20, and 9/20). Mice that survived the fourth injection often succumbed after a fifth injection. Because this toxicity looked very similar to the toxicity following immunization with the P815E peptide, we measured TNF levels in the serum of mice immunized either with chimeric peptide P1A-VIL1 or with peptide P1A. We observed higher and sustained levels of TNF in the mice immunized with P1A-VIL1, suggesting the involvement of TNF in the toxicity (Fig. 6Go). To see whether the toxicity was related to a massive expansion of specific CD8+ T cells, we used H-2Ld-P1A tetramers to stain PBL of immunized mice ex vivo. We observed an approximate 8-fold increase in the percentage of H-2Ld-P1A-positive cells among CD8+ cells in the blood of mice immunized with P1A-VIL1 as compared with mice immunized with P1A (Fig. 7Go). These results suggest that the linkage of the IL-1 peptide to the P1A peptide allowed a great expansion of specific CD8+ T cells, which results in TNF-mediated toxicity after repetitive immunizations. Our data also indicate that toxicity can occur with different antigenic peptides when massive CD8+ T cell responses are induced.



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  		FIGURE 7. Expansion of specific CD8+ T cells after immunization with peptide P1A-VIL1. Mice were immunized four times with either peptide P1A or chimeric peptide P1A-VIL1. Blood was collected from surviving mice 7 or 8 days after the last injection, and lymphocytes were stained with H-2Ld-P1A tetramers, with anti-CD8 and anti-CD3 Abs. Each • represents the percentage of tetramer-positive cells among CD8+ lymphocytes of an individual mouse. All tetramer-positive cells were CD8+. The average percentage of CD8+ among CD3+ cells was 20.9 in P1A-VIL1-immunized mice and 9.7 in P1A-immunized mice. Immunizations were performed in the presence of adjuvant in the absence of IL-12. Control mice received four injections of PBS with adjuvant.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The toxicity reported here appears to result from TNF produced by specific CD8+ T cells which were enormously expanded in mice immunized with peptide P815E. The immunizations were performed by footpad injections of peptide mixed with a QS21-based adjuvant and IL-12 according to a protocol that we had optimized to induce good CTL responses with various tumor antigenic peptides (9). Mice receiving only the adjuvant and IL-12 at the third injection showed a transient peak of circulating TNF (Fig. 2Go), which might be due to MPL, one of the components of the adjuvant, which is known to trigger TNF production by macrophages (31). However, circulating TNF in those mice quickly dropped to basal levels and did not cause toxicity. Therefore, neither the adjuvant nor the IL-12 was directly responsible for the toxicity, which was also triggered when the third injection was performed with peptide alone. The presence of the adjuvant and IL-12 during the first two injections did not cause immediate toxicity, but allowed the induction of very large numbers of specific CD8+ T cells.

Toxicity was also observed in mice immunized with other peptides provided a strong expansion of specific CD8+ T cells was achieved. For the P1A peptide, this required the adjunction of an IL-1 sequence to the peptide (Figs. 6Go and 7Go). For peptide P198, in experiments not reported here, we observed toxicity in some mice after numerous (more than six) repetitive immunizations with peptide/adjuvant/IL-12 which induced strong CTL responses. Nevertheless, it appears that an expansion of specific CD8+ lymphocytes occurs much more readily after immunization with peptide P815E than with other peptides. The reason for this is unclear. It could be speculated that in a normal immune response, a number of regulatory mechanisms are responsible for maintaining the CD8+ response within certain limits and that, for some reason, such mechanisms would be less active or even ineffective to control the response to peptide P815E. In this respect, it is interesting to note that the CD8+ response to peptide P815E is CD4+ independent (Fig. 3GoB) as opposed, for example, to the response to the P1A peptide, which is known to be CD4+ dependent (Ref. 32 and data not shown). Involvement of regulatory mechanisms could also explain why a large expansion of CD8+ T cells was not observed when mice were immunized with L1210.P1E.B7-1 cells instead of peptide P815E. The L1210.P1E.B7-1 cells may trigger such regulatory mechanisms more efficiently than the peptide, e.g., through B7-1-CTLA4 interactions.

Toxicity after peptide immunization has not been reported before. The only undesired phenomenon described is the induction of T cell unresponsiveness after injection of mice with high doses of peptide (500 µg) (33, 34). Injected at this dose into naive mice, a lymphocytic choriomeningitis virus peptide was found to induce specific T cell tolerance, presumably resulting from presentation of the peptide to naive T cells by nonprofessional APC (35). When the same peptide was injected at the same dose into lymphocytic choriomenigitis virus-immune mice, general immunosuppression associated with spleen damage was observed. This nonfatal spleen immunopathology was believed to result from the destruction of peptide-loaded spleen cells by Ag-specific CTL in vivo and was shown to be dependent on cell-cell contact and therefore not mediated by cytokines (35). The toxicity we report here is different from this immunopathology, because it is lethal and clearly mediated by a cytokine.

Our results indicate that peptide immunization can be dangerous in certain circumstances, since very strong CTL responses can be induced and can result in cytokine-mediated toxicity. Caution should be taken however in extrapolating our observations to the human situation in view of the very large difference in the peptide dose used relative to the size of the organism. We have used 50 µg of peptide for each injection in mice. In human peptide vaccination trials, the dose of peptide injected is typically between 30 µg and 1 mg (36, 37), whereas the human body volume is ~3000 times larger than that of a mouse. We have tried to reduce the peptide dose in our mouse immunizations, and this clearly resulted in reduced incidence of toxicity. But it also reduced the induction of CTL (data not shown). This suggests that there is a minimal amount of peptide required for efficient induction of T cells locally, either at the vaccine site or in the draining lymph node. Because there is no reason to believe that this "local" threshold is different in mice and humans, it is possible that a dose of 50 µg of a putative human peptide with properties similar to peptide P815E would induce good T cell responses in humans. Yet toxicity could be avoided because an expansion of CD8+ T cells to the same absolute numbers would result in lower proportions of specific CD8+ as they would be diluted among 3000 times more lymphocytes than in a mouse. Similarly, the same amount of TNF secreted by those expanded CD8+ T cells would be diluted in a much larger volume of body fluid. On the other hand, it is not impossible that humans are more sensitive to the toxicity of TNF than mice. For all of these reasons, it is difficult to extrapolate our observations to the human situation, but our results nevertheless suggest that new peptide immunization trials should be applied with caution in humans, using escalating dose regimens of the different vaccine components combined with a careful clinical surveillance and evaluation of the T cell response, including a monitoring of the expansion of specific CD8+ T cells with tetramers or similar reagents.


   Acknowledgments
 
We thank Sofia Buonocore, Dominique Donckers, and Luc Pilotte for their precious assistance, Dr. Guy Warnier for providing mice, Dr. Vincent Stroobant for peptide synthesis, Dr. Sylvie Cayphas for preparing the adjuvant, and Dr Jacques Van Snick for helpful discussions. We also thank Suzanne Depelchin for help in the preparation of this manuscript.


   Footnotes
 
1 This work was supported in part by a grant from the FB Assurances and VIVA (Belgium) and by Grants QLG1-CT-1999-00622 and QLK3-CT-1999-00064 from the Fifth Framework program of the European Community. J.B. was supported by a fellowship from the Christian de Duve Institute of Cellular Pathology (Brussels, Belgium). Back

2 J.B. and C.U. contributed equally to this work. Back

3 Current address: Immunex Corporation, 51 University Street, Seattle, WA 98101. Back

4 Address correspondence and reprint requests to Dr. Benoit J. Van den Eynde, Ludwig Institute for Cancer Research, 74 Avenue Hippocrate UCL 74.59, B-1200 Brussels, Belgium. E-mail address: benoit.vandeneynde{at}bru.licr.org Back

5 Abbreviations used in this paper: MPL, monophosphoryl lipid A; m, murine. Back

Received for publication April 4, 2002. Accepted for publication July 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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