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IL-12 Administration Leads to a Transient Depletion of T Cells, B Cells, and APCs and Concomitant Abrogation of the HLA-A2.1-Restricted CTL Response in Transgenic Mice

Katrin Peter^*, Michael J. Brunda and Giampietro Corradin^1,*

^* Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland; and Department of Oncology, Hoffmann-LaRoche, Nutley, NJ 07110

	Abstract

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The injection of a mixture of bona fide T cell epitopes can lead to the occurrence of immunodominance, meaning that the immune response is focused on the recognition of a single epitope or a small portion of the epitopes injected. We have previously demonstrated that the administration of rIL-12 can counteract immunodominance in BALB/c mice. In this study, we show that the administration of rIL-12 to HLA-A2.1 transgenic mice (A2k^b mice) abrogates specifically the immune response against HLA-A2.1-restricted HIV epitopes in the spleen. This lack of immune response is most probably due to a transient depletion of B cells, T cells, macrophages, and dendritic cells in this organ. Therefore, our study explains the mechanism of immunosuppression by rIL-12 in vivo.

	Introduction

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The development of successful vaccines requires the inclusion of a mixture of epitopes for the induction of an effective immune response that can protect against pathogens with high mutation rates, such as HIV. In addition, the incorporation of several epitopes restricted to different MHC haplotypes is necessary to cover the genetic differences of the population. Unfortunately, the application of such vaccine cocktails can lead to the occurrence of immunodominance (ID),² indicating that the immune response is limited to one epitope or a small portion of the bona fide T cell epitopes injected (1, 2, 3, 4). ID can therefore critically hinder the development of an effective and broad immune response against pathogens after immunization with an epitope mixture. We have previously reported that the administration of rIL-12 during 5 consecutive days initiated before immunization with CTL epitopes could overcome ID in BALB/c mice (5). In the present study, we tested the application of rIL-12 in the immunization of HLA-A2.1 transgenic mice (A2k^b mice) (6) with HIV epitopes restricted to the HLA molecule. These epitopes induced a good immune response when injected individually. However, the immune response against some of the epitopes was abrogated when they were injected as a mixture (7). Unexpectedly, the rIL-12 regimen led to a complete abrogation of the immune response to these peptides. This abrogation was specific for the HLA-A2.1-restricted peptides, but not for the K^b-restricted epitopes. Our data suggest that the lack of immune response is related to and possibly the consequence of a transient depletion of B cells, T cells, macrophages, and dendritic cells in the spleen.

	Materials and Methods

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Peptides

The following peptides were synthesized by the F-moc, t-Bu strategy for solid phase systems, as described previously by Atherton et al. (8): Influenza matrix protein MA 58–66 (flu); tetanus toxoid peptide tt 947–967 (P30); and HIV peptides, RT 476–484 (LR22), p17 77–85 (LR23), RT 346–354 (LR26), gp41 814–823 (LR27), and RT 956–964 (LR28). Peptides were purified with a G25 size exclusion chromatography and further subjected to mass spectrometric analysis (purity >80%). The melanoma peptide TRP2 was kindly provided by Dr. P. Romero (Ludwig Cancer Institute, Epalinges, Switzerland).

Animals

The HLA-A2.1 transgenic mouse strain (A2k^b) was produced by Irwin et al. (9) and was kindly provided by Dr. P. Romero. Male or female mice were used at the age of 6–12 wk.

Cell culture

EL-4/HLA-A2.1 cell line. EL-4 cells transfected with the HLA-A2.1 class I molecule were used for CTL assays and for in vitro restimulation of CTL cultures. Cells were cultured in DMEM with Glutamax supplemented with 10 mM HEPES (Life Technologies, Paisley, U.K.), 10% FCS (Seromed, Biochrom KG, Berlin, Germany), and 600 µg/ml G418 (Calbiochem, La Jolla, CA).

Vaccine formulations, immunization, and IL-12 treatment

Stock solutions of HIV peptides and flu peptide at concentrations of 10 mg/ml were prepared in DMSO. A stock solution of P30, which served as a universal Th epitope (10, 11), was prepared in H₂O at a concentration of 10 mg/ml. The vaccine formulations were prepared in IFA (Difco Laboratories, Detroit, MI): the HIV peptides and P30 were diluted in PBS to concentrations of 1 and 2 mg/ml, respectively. Peptide solutions were mixed with IFA at a ratio of 1:1 and sonicated until a sticky emulsion was obtained. Mice were immunized with 50 µl s.c., corresponding to 25 µg CTL epitope and 50 µg Th epitope per mouse.

IL-12 was a Chinese hamster ovary cell-derived mouse recombinant protein (12) and produced by Hoffmann-LaRoche (Nutley, NJ). Mice were injected with rIL-12 i.p. five times for 5 consecutive days with 1 µg rIL-12 in 100 µl PBS starting from day -1 relative to immunization. The same regimen was applied for the evaluation of the K^b and K^d expression in C57BL/6 and BALB/c mice. However, mice were not immunized, because the down-regulation of MHC class I molecules by IL-12 could be observed independently of injection with peptides.

In vitro CTL stimulation

Spleen was removed 10 days after immunization, and single cell suspensions were prepared. Spleen cells were cultured in six-well plates at 3 x10⁷ cells/well together with 1 µM peptide. The culture medium was DMEM with Glutamax supplemented with 10% FCS, 10 mM HEPES, and 50 µM 2-ME (Merck-Schuchardt, Hohenbrunn, Germany). Separate cultures were set up for each individual CTL epitope. For restimulation, CTLs were cultured in a 24-well plate at a density of 5 x 10⁵ cells/well together with 2 x 10⁵ EL-4/A2.1 cells that were pulsed with 1 µM peptide for 1 h at 37°C, washed, and irradiated with 10 krad, and 5 x 10⁶ spleen cells from a syngeneic mouse that were washed and irradiated with 5 krad. The culture medium was DMEM with Glutamax supplemented with 10% FCS, 10 mM HEPES, 50 µM 2-ME, and 30 U/ml EL-4 culture supernatant as a source of IL-2.

CTL assay

The CTL activity of the cell cultures was determined after 7 days of in vitro stimulation in a standard ⁵¹Cr release assay. Briefly, 5 x 10⁵ HLA-A2.1-transfected EL-4 cells were labeled for 1 h at 37°C with 50 µCi (⁵¹Cr) chromate in the presence of 1 µM CTL epitope. After three washes, 1 x 10³ labeled cells were added to serial dilutions of spleen cells in V-bottom microtiter plates in a final volume of 200 µl. The medium was DMEM supplemented with 5% FCS and 10 mM HEPES. After 4 h of incubation at 37°C, the supernatants (100 µl) were harvested for gamma counting. The percent lysis was calculated as: 100 x ((experimental - spontaneous release)/total - spontaneous release)).

Staining of surface molecules on spleen cells

Abs used: anti-Fc II (2.4G2), biotinylated anti-HLA-A2.1 (BB7.2), biotinylated anti-K^b (AF6-88.5), Streptavidin-CyChrome, FITC anti-K^d (SF-1-1.1), FITC anti-CD45R/B220 (RA3-6B2), FITC anti-CD3 (17A2), FITC anti-CD11c (HL3), PE anti-I-A/I-E (M5/114.15.2), PE anti-Ly-6D/GR-1) (RB6-805), PE anti-TER119 (TER119), PE anti-pan-NK (DX5), PE anti-CD19 (1D3), PE anti-CD44, PE anti-CD62L, and PE anti-CD138/Syndecan-1 (281-2). All Abs were obtained from BD PharMingen (San Diego, CA), except the anti-CD44 and anti-CD62L that were obtained from Caltag Laboratories (South San Francisco, CA), and the anti-Fc that was kindly provided by Dr. J. Louis (WHO-IRTC, Biochemistry Institute, Epalinges, Switzerland). The anti-HLA-A2.1 Ab was produced from the American Type Culture Collection (Manassas, VA) hybridoma cell line HB-82.

Cells were incubated with the anti-Fc II Ab in PBS 1% BSA (Fluka Chemika, Buchs, Switzerland) for 20 min at 4°C. Cells were washed twice with PBS/1% BSA and again incubated with the specific Abs for 30 min at 4°C in PBS/1% BSA. If staining was performed with biotinylated Abs, cells were washed twice with PBS/1% BSA and incubated with streptavidin CyChrome for 15 min. After washing with PBS/1% BSA, cells were analyzed with a (FACScan; BD Biosciences, San Jose, CA).

	Results

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The administration of rIL-12 to A2k^b mice abrogates the immune response against HLA-A2.1-restricted HIV peptides

We have previously shown that the injection of BALB/c (5) and A2k^b mice (7) with a mixture of peptides resulted in a weaker immune response than that observed if the peptides were injected individually. These data have been explained by the establishment of ID. In BALB/c mice, ID could be overcome with the administration of rIL-12 before injection with CTL epitopes (5). According to this protocol, we treated A2k^b mice with rIL-12 (1 µg/day/mouse) during 5 consecutive days starting at day -1 before immunization with five HIV peptides. The spleen was removed 10 days after immunization. The rIL-12 treatment induced a significant enlargement of the spleen, as has been reported earlier (5, 13). The immune response was evaluated 1 wk after in vitro stimulation using a standard ⁵¹Cr release assay. All the peptides were able to elicit an immune response, although not to the same extent (Fig. 1A). Surprisingly, however, the immune response in these mice was totally abrogated after IL-12 administration for the peptides LR22, LR23, LR27, and LR28 (Fig. 1B). In contrast, the immune response against LR26, a peptide that is restricted to the endogenous murine MHC class I molecules, was maintained.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Immunization of mice, with or without IL-12 pretreatment, with a mixture of peptides. A, A2kb mice were immunized with 25 µg of each peptide together with 50 µg P30 in IFA. Mice were sacrificed 10 days after injection, and specific lysis was evaluated 1 wk after in vitro stimulation. HLA-A2.1-transfected EL-4 cells were used as target cells. B, Mice were injected during 5 consecutive days with IL-12 (1 µg/day) starting on day -1 before immunization with the peptide mixture. As in A, mice were sacrificed after 10 days, and specific lysis was evaluated after 7 days of in vitro stimulation. HLA-A2.1-transfected EL-4 cells were used as target cells. For further details, see Materials and Methods.

We investigated whether this abrogation of the immune response was specific for HLA.A2.1-restricted peptides. For this purpose, we immunized A2k^b mice with either 25 µg HLA-A2.1-restricted flu peptide ± rIL-12 or 25 µg K^b-restricted melanoma peptide TRP2 ± rIL-12. The immune response against the flu peptide was abrogated in rIL-12-treated mice, whereas it was normal for TRP2, indicating that the abrogation is limited to HLA-A2.1-restricted peptides (Fig. 2). Moreover, it indicates that IL-12 did not have a general immunosuppressive effect. The loss of the immune response against the HLA-A2.1-specific flu peptide could not be observed at lower doses of rIL-12 (data not shown). In contrast, the enlargement of the spleen that is normally associated with the rIL-12 administration could not be observed at lower doses.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Immunization of mice, with or without IL-12 pretreatment, with the flu or TRP2 peptide. Mice were injected during 5 consecutive days with IL-12 (1 µg/day) starting on day -1 before immunization with 25 µg of either flu or TRP2 peptide together with 50 µg P30 in IFA. Mice were sacrificed 10 days after injection, and specific lysis was evaluated 1 wk after in vitro stimulation. HLA-A2.1-transfected EL-4 cells were used as target cells. For further details, see Materials and Methods.

Since the abrogation of the immune response after rIL-12 treatment is specific for HLA-A2.1-restricted peptides, we studied whether this was a result of a down-modulation of HLA-A2.1 molecules. Therefore, we evaluated the expression of the HLA-A2.1 molecule on splenocytes using FACS analysis. A2k^b mice were treated with rIL-12 and immunized with the flu peptide, as described above, and the expression of HLA-A2.1 on splenocytes was evaluated 10 days after immunization. HLA-A2.1 expression on 60% of the cells was reduced in rIL-12-treated mice. Similarly, the K^b expression was reduced on 65% of cells (Fig. 3A). These results could be observed independently of immunization with peptides (data not shown). Similar results were also obtained in C57BL/6 mice and in BALB/c mice in which the K^b and K^d expression on splenocytes was reduced, respectively (data not shown). We subsequently evaluated the expression of MHC class I molecules on B cells, T cells, macrophages, and dendritic cells isolated from the spleen at days 0, 3, 7, 10, 14, and 30 of rIL-12 injection using the B220, CD3, CD11c, and MHC class II surface markers to distinguish the various cell types. Interestingly, we found a depletion of B cells, T cells, and macrophages at days 7, 10, and 14 and a depletion of dendritic cells at day 14, which was accompanied with an accumulation of a cell population that was negative for the specific cell markers as well as for the MHC class I molecules (Table I). Fig. 3B shows the B220 and MHC class I expression on B cells on day 14 with or without treatment of rIL-12. The depletion was transient, and a normal cell population could again be observed 30 days after the first rIL-12 injection. Thus, the observed reduction of MHC class I on total splenocytes was rather due to a temporary change of the existing cell population than to an actual down-regulation of this molecule on the cell surface. Indeed, the remaining B cells, T cells, and APCs showed a similar or higher expression of MHC class I after rIL-12 injection as compared with controls (data not shown). Stainings performed for various cell surface markers such as Gr-1, Pan-NK, TER119, CD19, CD44, CD62L, and CD138 (Syndecan 1) demonstrated that the newly accumulated cell population did neither contain any granulocytes, which may point toward an inflammation, nor any NK cells, activated B cells, or plasma cells. However, they were CD44 positive and CD62L negative, and 55% of these cells showed an increase in TER119 (Fig. 4). This indicates that rIL-12, while having dramatic effects for most cell types, may at the same time induce erythropoiesis.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Evaluation of HLA-A2.1 and Kb expression on total splenocytes and on spleen B cells. A, A2kb mice were treated during 5 consecutive days with 1 µg rIL-12 per day, and the expression of HLA-A2.1 and Kb on total splenocytes was evaluated 14 days after the first injection. B, A2kb mice were treated during 5 consecutive days with 1 µg IL-12 per day, and the expression of HLA-A2.1 and Kb on B cells derived from the spleen was evaluated 14 days after the first injection. For further details, see Materials and Methods.

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Evaluation of TER119 expression on spleen B cells with or without IL-12 treatment in A2kb mice. A2kb mice were treated during 5 consecutive days with 1 µg rIL-12 per day, and the expression of TER119 on B cells derived from the spleen was evaluated 14 days after the first injection. For further details, see Materials and Methods.

	Discussion

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While early reports stressed on the role of IL-12 on the potentiation of the immune responses (20, 21), in the recent years, suppressive effects of IL-12 have been observed (22, 23, 24, 25). It has been proposed that the immune suppression by high doses of IL-12 was due to the induction of IFN- production by host lymphocytes. In turn, IFN- would activate macrophages and induce inducible NO synthase activity, which would generate NO production that impairs the proliferation of T cells in response to mitogens (23). However, whether in our case iNOS activity may be responsible for the depletion of B cells, T cells, and APCs and the subsequent abrogation of the immune response remains to be evaluated.

It is intriguing to observe that only the HLA-A2.1-specific response is abrogated, although both the number of HLA-A2.1 and K^b-expressing cells is reduced. In contrast, in the HLA-A2.1 transgenic mice, only 50% of splenocytes express the HLA-A2.1 molecule at 40% of the level of the endogenous MHC class I molecules (6). It is therefore plausible that the abrogation of the HLA-A2.1-specific immune response may reflect the depletion of the HLA-A2.1-expressing cells below a critical threshold for the presentation of Ag. For the K^b- and K^d-restricted peptides, this diminution does not reach that critical level, and presentation does occur. In this case, the beneficial effect of rIL-12 can be observed.

In this study, we have demonstrated that rIL-12 can have dramatic immunoregulatory effects associated with a depletion of B cells, T cells, and APCs, leading to an abrogation of the immune response in HLA-A2.1 transgenic mice. This is in contradiction to reports that IL-12 may be an excellent candidate for use as an immunopotentiator, especially for cell-mediated immune responses. We have observed that lower doses of IL-12 did not have this suppressive effect on the HLA-A2.1-specific immune response. In contrast, the enlargement of the spleen that is normally associated with the rIL-12 administration could not be observed at lower doses. The application of rIL-12 as adjuvant for vaccination therefore needs further careful evaluation. In addition, the previously reported effect of rIL-12 on ID in BALB/c mice (5) needs to be reexamined to determine the mechanism of ID modulation.

	Acknowledgments

 
We thank Dr. Heike Voigt for technical help with the FACS analysis, and Dr. Daniela Finke for scientific discussions and critical reading of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Giampietro Corradin, Institute of Biochemistry, Ch. des Boveresses 155, University of Lausanne, Epalinges, Switzerland. E-mail address: giampietro.corradin{at}ib.unil.ch

² Abbreviation used in this paper: ID, immunodominance.

Received for publication April 20, 2001. Accepted for publication April 26, 2002.

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