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The Journal of Immunology, 2006, 177: 306-314.
Copyright © 2006 by The American Association of Immunologists

IL-2 In Vivo Activities and Antitumor Efficacy Enhanced by an Anti-IL-2 mAb1

Daisuke Kamimura*,{ddagger}, Yukihisa Sawa{dagger}, Masae Sato{ddagger}, Eviryanti Agung{dagger}, Toshio Hirano*,{dagger},{ddagger} and Masaaki Murakami2,{ddagger}

* Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan; {dagger} Department of Developmental Immunology, Osaka University Graduate School of Frontier Biosciences, Yamadaoka, Suita, Osaka, Japan; and {ddagger} Department of Developmental Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 cDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8+ T and NK1.1high cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44highCD8+ population, and the increased population was maintained for >300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin 2 is a cytokine produced mainly by activated T cells and a subset of CD4+ T cells under normal conditions and is a potent growth factor for immune cells, including T cells and NK cells, in vitro (1, 2). Because treatment with exogenous IL-2 shows immunostimulatory effects in animals and humans, IL-2 immunotherapy has long been tested for clinical applications (1). However, the accumulated evidence suggests that the endogenous level of IL-2 has rather immunosuppressive and autoimmune-preventing activities in vivo (3). A recent study formally demonstrated, using foxp3gfp mice, that IL-2 is a critical survival factor for Foxp3+-regulatory CD4+ T cells (4), which explains the development of autoimmune disorders in IL-2 knockout (KO),3 CD25 KO, and CD122 (the common subunit for IL-2R and IL-15R) KO mice (5, 6, 7). The disease states associated with lymphoproliferation in these animals often hamper the acquisition of genetic evidence for the in vivo functions of IL-2 in various experimental systems. To overcome this, neutralizing and/or signal-blocking Abs against IL-2 and its receptor subunits have been used to investigate the in vivo IL-2 functions. One widely used neutralizing mAb is anti-murine IL-2 mAb clone S4B6 (8). This mAb is a rat IgG2a known to inhibit the activity of murine, but not human or rat, IL-2 (9). Because recombinant murine IL-2 (rmIL-2) derived from bacteria, yeast, monkey, and mouse cells are recognized by the mAb, the carbohydrate modification of IL-2 seems not to be important for the mAb binding (9). Extensive mutational studies revealed that an intact N-terminal portion of mIL-2 (Q26 to Y45) is required for its recognition by the anti-IL-2 mAb (9). This region encompasses an amino acid residue (D34) within the {alpha} helix A that is critical for the CD122 binding of IL-2 (10). In addition, the peptide encompassing {alpha} helix A of human IL-2, termed p1–30, acts as an agonist through CD122 (11). These lines of evidence establish the neutralizing potential of the anti-IL-2 mAb.

Using this anti-IL-2 mAb, we previously found that the division of memory CD8+ T cells increases significantly after IL-2 depletion in vivo (12, 13, 14). The increased cell divisions are associated with a reduction in the CD25+CD4+-regulatory T cells, due to the IL-2 depletion (13), are independent of IL-15 but require CD122 signaling in memory CD8+ T cells (14). The CD122 dependency in IL-2-depleted IL-15 KO mice led us to hypothesize that a novel CD122-targeted cytokine mediates the increased division of the memory CD8+ T cells (14). To clone this factor, we developed an in vivo screening system using a hydrodynamic plasmid DNA delivery method (15). In the course of developing this assay, however, we noticed that the anti-IL-2 mAb S4B6 unexpectedly seemed to enhance the biological activities of IL-2 in vivo.

Previous studies showed that Abs against human IL-2 (16, 17, 18, 19), human IFN-{alpha} (20), and murine IL-3, IL-4, IL-6, and IL-7 (21, 22, 23) act as carrier proteins for their respective cytokines, prolonging their serum half-life and in most cases enhancing their bioactivities.

Here we demonstrated that the widely used neutralizing anti-murine IL-2 mAb S4B6 also has IL-2 potentiating effects in vivo, especially when IL-2 is abundant. Furthermore, by exploiting this unexpected property, we found that concurrent treatment with the mAb and IL-2 gene delivery preferentially and significantly increased the numbers of cells belonging to types believed to function in tumor immunosurveillance, including NK1.1highTCRbeta– classical NK cells and NK1.1+CD44highCD8+ T cells (24, 25), and elicited effective antitumor immunity in mice.


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

C57BL/6J (B6) mice were purchased from CLEA Japan. B6.SJL (CD45.1) mice were obtained from Taconic. B6.OT-1 TCR-transgenic mice were provided by Dr. W. R. Heath. All strains were kept under specific pathogen-free conditions in the animal facility of RIKEN RCAI. The animal experiments were approved by the RIKEN Yokohama Institute Safety Center.

Reagents and mAbs for flow cytometry

The mAbs used for flow cytometry were: PE-carbocyanin (Cy)7- or allophycocyanin-labeled anti-CD4 mAb (RM4-5); PE- or PE-Cy7-labeled anti-CD8{alpha} mAb (53-6.7); PE-Cy7-labeled anti-CD19 mAb (6D5); FITC- or allophycocyanin-labeled anti-CD44 mAb (IM7); biotinylated anti-CD45.1 mAb (A20); PE-labeled anti-CD45R (B220) mAb (RA3-6B2); FITC-labeled anti-CD69 mAb (H1.2F3); PE-labeled anti-V{alpha}2 TCR mAb (B20.1); biotinylated anti-NK1.1 mAb (PK136); and FITC-labeled TCRbeta chain (H57-597). PE-Cy7- or allophycocyanin-conjugated streptavidin was used to detect the biotinylated mAbs. These mAbs and streptavidin reagents were obtained from BD Biosciences, eBioscience, BioLegend, and Caltag Laboratories. Foxp3+-regulatory T cells were detected using the allophycocyanin anti-mouse/rat Foxp3 Staining Set (eBioscience).

Preparation and injection of IL-2 plasmid DNA

The full-length cDNA for mIL-2 was obtained by PCR using cDNA from PMA-activated CD8+ T cells. The mIL-2 cDNA in the presence or absence of IL-2 tagged with 6x His at the C terminus (IL-2-His) was cloned into expression vectors: the pCAGGS vector (15); and Gateway pcDNA series destination vectors (Invitrogen). The controls were the Gateway vectors containing GFP or the pCAGGS vector without insert DNA. These plasmid DNAs were purified with the GenElute endotoxin-free plasmid Midiprep kit (Sigma-Aldrich) or EndoFree plasmid Mega kit (Qiagen). For IL-2 protein expression in vivo, mice were given a bolus injection of 2 ml of lactate Ringer’s solution containing the IL-2 plasmid DNA. The pCAGGS plasmid vector induces very strong protein expression through a combination of the CMV enhancer and chicken beta-actin promoter (15). In this study, this vector was used only for the serum IL-2 Western blot assay, because IL-2 from the pCAGGS vector, even when as little as 10 µg were given, was associated with mortality 4–5 days after the treatment, but up to 50 µg of pcDNA/mIL-2 did not have this effect. The dose of 6.25 µg of the pcDNA was determined by dose-response experiments (data not shown). Some mice were also given i.p. injections of 1 mg of anti-IL-2 mAb (S4B6) or rat IgG (Sigma-Aldrich). Preparation of the protein G-purified anti-IL-2 mAb and anti-CD122 mAb (TMbeta1) was performed as described previously (14). A sandwich ELISA system for IL-2 was obtained from BioSource International.

Preparation of the anti-IL-2 mAb/rIL-2 mixture

The anti-IL-2 mAb or rat IgG (1 mg) and rmIL-2 or recombinant human IL-2 (rhIL-2; 2 µg; PeproTech) were mixed in vitro and incubated overnight at 4°C with rotation. After the incubation, the IL-2 signal in the mixture containing the anti-IL-2 mAb and rmIL-2 was not detectable by the IL-2 ELISA, suggesting that an immune complex had formed. These mixtures were injected i.p. into wild-type (WT) mice. Some mice were also given 200 µg of anti-CD122 mAb (TMbeta1) by i.p. injection. Cell turnover was assessed after 5 days by in vivo BrdU labeling.

In vivo BrdU labeling

Mice were given 0.8 mg/ml BrdU (Sigma-Aldrich) in their drinking water during the treatment. Intracellular BrdU staining was performed using the BrdU Flow kit (BD Biosciences).

Western blotting analysis of serum IL-2

The serum (0.1 µl) from mice that had been treated with the pCAGGS vectors including a empty control or the vectors carrying IL-2, or IL-2-His (50 µg/mouse, i.v.) and Abs 1 day before was separated by reducing or nonreducing SDS-PAGE and blotted onto a polyvinylidine difluoride membrane (Immobilon; Millipore). After the blocking of nonspecific protein binding, the membrane was probed with the HRP-conjugated anti-IL-2 mAb (S4B6), which was prepared using the Peroxidase Labeling kit-NH2 (DOJINDO), or goat anti-mouse IgG (H+L)-HRP (Zymed). The HRP signal was revealed using ECL procedures.

For Western blotting to identify the complex between anti-IL-2 Ab and IL-2 in serum, the serum (1 µl) was mixed with a standard 1x SDS sample buffer (99 µl; with or without 2-ME) and heated for 10 min for the reducing condition and left at room temperature for 10 min for the nonreducing condition. The resulted samples were run on 2–15% PAGE gel (Dai-ichikagaku). Western blotting was performed by a standard method using anti-His-tag Ab.

In vivo cytotoxicity assay

The lymph nodes (inguinal, axillaries, cervical, and mesenteric) and spleen were harvested from OT-1 TCR transgenic mice, passed through a 100-µm pore size cell strainer (BD Falcon) with RPMI 1640, and washed. Erythrocytes were eliminated with 0.165 M NH4Cl. A T cell-enriched sample was then prepared using a nylon wool column. The purified cells containing 6 x 106 TCRV{alpha}2+CD8+ T cells were injected i.v. into WT B6 mice on day –2. These mice were then given a plasmid DNA injection (6.25 µg/mouse i.v.) with or without the anti-IL-2 mAb (1 mg/mouse i.p.) on day 0. Splenocytes from B6.SJL mice were incubated for 30 min at 37°C with or without 10 µg/ml OVA257–264 peptide (SIINFEKL; Operon Biotechnologies). These peptide-pulsed target and nontarget populations were labeled with 2.5 and 0.25 µM CFSE (Invitrogen Life Technologies), respectively, mixed at a 1:1 ratio, and injected i.v. into the mice. After 12 h, the target cell-killing activity was evaluated using the ratio of CFSE-positive populations in the spleen. The CFSE-labeled populations were distinguished from host populations by CD45.1 expression.

B16 lung metastasis model

B16 melanoma cells were kindly provided by Dr. Shin-ichiro Fujii (RIKEN RCAI). B16 cells (2 x 105/mouse) were injected i.v. on day 0. The tumor-bearing mice received a plasmid DNA injection (6.25 µg/mouse i.v.) with or without the anti-IL-2 mAb (1 mg/mouse, i.p.) on day 2. The number of B16 nodules in the lungs was counted on day 14.

Statistical analysis

Statistical differences between groups were examined by unpaired Student’s t tests. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Neutralizing anti-murine IL-2 mAb (S4B6) enhances the in vivo effect of IL-2

We have reported that in vivo treatment with the neutralizing anti-IL-2 mAb (clone S4B6) promotes the division of memory CD8+ T cells in mice, by an IL-15-independent but CD122-dependent mechanism (12, 14). To further investigate the in vivo roles of IL-2 and IL-15 and to screen for a putative novel CD122-targeted cytokine (14), we used an in vivo hydrodynamic delivery system for plasmid DNA, by which proteins of interest can be transiently expressed and their functions examined (15, 26). In the course of this assay, however, we unexpectedly found that the anti-IL-2 mAb potentiates the in vivo effects of IL-2.

After an i.v. injection of 6.25 µg of pcDNA expression vectors encoding murine IL-2 cDNA, serum IL-2 was transiently detected by ELISA; it returned to baseline by 3 days posttreatment (see Fig. 1A, {circ}). As expected, overexpression of IL-2 by the IL-2 plasmid (plus rat IgG) significantly increased the BrdU uptake in CD8+ T cells in vivo, as compared with the vehicle injection (Fig. 2A). Total splenocyte counts also increased 5 days after the IL-2 plasmid DNA injection (Fig. 2B, hatched columns).


Figure 1
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  		FIGURE 1. The anti-IL-2 mAb increases and sustains serum IL-2 levels upon IL-2 plasmid DNA infusion. A, Serum IL-2 levels after the injection of plasmid DNAs (6.25 µg) with or without the anti-IL-2 mAb, measured by ELISA. Data are given as the mean ± SEM (n = 4–5). *, p < 0.01 vs treatment with rat IgG and the control plasmid. B, Serum IL-2 (top) and L-chain of IgG as the loading control (bottom) 1 day after treatment were detected by Western blotting. Lane 1, rat IgG plus pCAGGS; lane 2, anti-IL-2 mAb plus pCAGGS; lane 3, rat IgG plus pCAGGS/mIL-2; lane 4, anti-IL-2 mAb plus pCAGGS/mIL-2. The doses of Abs and plasmid DNA were 1 mg and 50 µg, respectively.

 

Figure 2
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  		FIGURE 2. An anti-IL-2 mAb, clone S4B6, enhances the in vivo effects of IL-2 delivered as an IL-2 plasmid DNA injection. A, Mice were treated i.v. with lactate Ringer’s solution (vehicle) or 6.25 µg of pcDNA plasmid carrying mIL-2 cDNA (IL-2 plasmid) and given an i.p. injection of 1 mg of rat IgG or the anti-IL-2 mAb at the same time. The mice were given BrdU in their drinking water for 3 days. BrdU levels in the spleen were examined on day 4. Plots are gated on the TCRbeta+CD8+ population. B, Absolute numbers of splenic subpopulations were counted 5 days after the combined plasmid DNA and mAb treatment. {square}, Rat IgG plus control plasmid DNA; {cjs2108}, anti-IL-2 mAb plus control plasmid DNA; {cjs2113}, rat IgG plus IL-2 plasmid DNA; {blacksquare}, anti-IL-2 mAb plus IL-2 plasmid DNA. Each column indicates the mean ± SEM (n = 3–5). *, Statistical significance vs treatment with rat IgG and the control plasmid. *, p < 0.05; **, p < 0.01).

 
To check whether this effect was really due to IL-2 overexpression, the anti-IL-2 mAb (S4B6), which has been used as a neutralizing mAb, was injected i.p. at the same time as the IL-2 plasmid DNA. As reported previously, the anti-IL-2 mAb (plus control plasmid DNA) increased the BrdU+CD44highCD8+ T cell population, compared with rat IgG and vehicle treatment (14). After the coinjection of the anti-IL-2 mAb and IL-2 plasmid DNA, however, the biological effects of IL-2 were markedly enhanced: almost all the CD8+ T cells became BrdU positive (Fig. 2A), and the number of splenocytes substantially increased compared with their number after treatment with rat IgG and IL-2 plasmid DNA (Fig. 2B). The preferential increase of CD8+ T cells and TCRbeta–NK1.1high NK cells accounted for most of the cell population expansion following the coinjection of the anti-IL-2 mAb and IL-2 plasmid DNA (Fig. 2B). These results suggest that the neutralizing anti-IL-2 mAb, clone S4B6, unexpectedly enhances the biological activities of IL-2 when it is overexpressed by injected plasmid DNA.

Anti-IL-2 mAb increases the amount of IL-2 in the serum

To investigate the effect of the anti-IL-2 mAb on serum IL-2 levels after IL-2 plasmid DNA injection, we first attempted to use an ELISA system. As shown in Fig. 1A, increased and sustained levels of serum IL-2 were detected after the anti-IL-2 mAb plus IL-2 plasmid DNA treatment compared with the rat IgG plus IL-2 plasmid DNA treatment. However, we found that a preformed complex of the anti-IL-2 mAb and rmIL-2 was not detected by the IL-2 ELISA (data not shown). This suggests that the mAb interfered with the ELISA system, thereby leading to an underestimation of the serum IL-2 levels attained after the anti-IL-2 mAb plus IL-2 plasmid combination treatment. Therefore, we sought to verify the ELISA results by a Western blotting analysis using sera from treated mice. No detectable signal corresponding to mIL-2 was obtained from the IL-2 plasmid DNA alone, when 0.1 µl of the serum was assayed (Fig. 1B, lane 3). In contrast, serum IL-2 was clearly detected by Western blotting after coinjection of the anti-IL-2 mAb and IL-2 plasmid (Fig. 1B, lane 4). The difference in signal intensity for serum IL-2 between the anti-IL-2 mAb plus IL-2 plasmid DNA and rat IgG plus IL-2 plasmid DNA on the Western blot was much greater than the ELISA results indicated (Fig. 1A and data not shown), suggesting that both free IL-2 and a relatively large amount of mAb-bound IL-2, which was not detectable by ELISA, were present in the circulation after the anti-IL-2 mAb plus IL-2 plasmid DNA injections.

Preformed complex of anti-IL-2 mAb and rmIL-2 also shows the agonist effect

We were concerned that the enhancement of the IL-2 bioactivity by the anti-IL-2 mAb might be observed only when the plasmid DNA infusion system was used. To exclude this possibility, 2 µg of rmIL-2 or rhIL-2, as a nonbinding control, was incubated in vitro with excess anti-IL-2 mAb (1 mg) to allow the formation of the immune complex (in the case of rmIL-2). After the in vitro incubation, the murine IL-2 ELISA failed to detect signals in the anti-IL-2 mAb and rmIL-2 mixture, suggesting that the immune complex had formed and was interfering with the ELISA system (data not shown). These mixtures were then injected into mice. As shown in Fig. 3, BrdU uptake in the CD44highCD8+ (Fig. 3, top) and NK1.1high (Fig. 3, bottom) populations was slightly increased by the coinjection of rat IgG and 2 µg of rmIL-2 or rhIL-2. The anti-IL-2 mAb treatment without rIL-2 (medium) enhanced BrdU uptake, as reported (14). The combination of the anti-IL-2 mAb plus rhIL-2 showed only an additive effect, particularly on the CD44highCD8+ population. In contrast, a synergistic effect was observed after treatment with the preformed complex of the anti-IL-2 mAb plus rmIL-2, and almost all the CD44highCD8+ and NK1.1+ cells became BrdU positive. This synergistic effect was diminished when an anti-CD122 mAb was injected to inhibit IL-2 signaling (Fig. 3). This result, together with the those shown in Fig. 2, demonstrate that the anti-IL-2 mAb enhances the biological activities of IL-2 in vivo, that this effect is mediated at least in part through IL-2Rbeta molecules (CD122), and that proper mAb/Ag recognition is required, given that the mAb plus rhIL-2 had only an additive and no synergistic effect.


Figure 3
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  		FIGURE 3. Preformed complex of the anti-IL-2 mAb and rmIL-2 is more effective than rmIL-2 alone. Anti-IL-2 mAb or rat IgG (1 mg) was incubated overnight in vitro with 2 µg of rmIL-2 or rhIL-2. Mice were given an i.p. injection of the mixture and BrdU in drinking water for 4 days. One treatment group also received an i.p. injection of anti-CD122 mAb (200 µg/mouse). BrdU levels in the spleen were assessed on day 5. Histograms are gated on the CD44highCD8+ (top) or the NK1.1high (bottom) population. The limit bars show percentage of BrdU+ cells.

 
The anti-IL-2 mAb reduces the Foxp3+-regulatory CD4+ T cell population in vivo

In contrast to the results shown in Figs. 2 and 3, i.p. injections of the anti-IL-2 mAb alone decreased the population of Foxp3+-regulatory CD4+ T cells after 7 days, by about one-half (Fig. 4), which is consistent with other reports using the same clone (2, 13). Moreover, the population of Foxp3+-regulatory CD4+ T cells in the periphery is about one-half that of WT in IL-2 KO and CD25 KO mice (4). Our result suggests that the anti-IL-2 mAb exhibits an in vivo neutralizing activity, at least for Foxp3+-regulatory CD4+ T cells, unless IL-2 is overexpressed or the protein level of IL-2 exceeds the physiological concentration.


Figure 4
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  		FIGURE 4. The anti-IL-2 mAb reduces the Foxp3+-regulatory CD4+ T cell population in vivo. Mice received an i.p. injection of 1 mg of anti-IL-2 mAb or rat IgG on days 0, 1, 2, 3, and 5. The Foxp3+ population in the spleen was examined on day 6. Data are from two mice for each treatment; the histograms are gated on the TCRbeta+CD4+ population. The limit bars show percentage of Foxp3+ cells.

 
Treatment with the anti-IL-2 mAb alone causes a long term increase in the CD44highCD8+ T cell population

Given our results thus far and the reported evidence that IL-2 is produced by a subset of CD4+ T cells under normal conditions (2), we were attracted to the idea that the anti-IL-2 mAb injected in vivo could increase IL-2 biological activity by capturing and retaining endogenous IL-2, which would then accumulate and eventually show increased activity. To investigate this possibility, WT mice were treated with the anti-IL-2 mAb alone for the first 5 days, and the CD44highCD8+ T cell population in the peripheral blood was checked periodically for >300 days. As shown in Fig. 5, the CD44highCD8+ T cell population in the anti-IL-2 mAb-treated mice gradually increased for ~50 days, and this increased CD44highCD8+ population could be detected for >300 days (Fig. 5, experiment 1). At this point, the control mice also showed a large proportion of CD44highCD8+ T cells due to the age-associated increase of these cells (12, 27). Similar results were obtained in a second set of experiments, in which rat IgG was used as a control (Fig. 5, experiment 2). These results indicate that the anti-IL-2 mAb may indeed capture the endogenous IL-2 and increase the IL-2 levels to exhibit enhanced biological activity in long term assays.


Figure 5
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  		FIGURE 5. Treatment with the anti-IL-2 mAb alone increases the CD44highCD8+ T cell population over time. Mice were given an i.p. injection of 1 mg of anti-IL-2 mAb or rat IgG for the first 5 days. Control groups had no treatment (experiment (Exp.) 1) or were given 1 mg of rat IgG on the same days (experiment 2). The PBL were examined periodically for CD44 levels on the CD8+ T cells. Data are from two mice for each treatment; histograms are gated on the CD8+ population. The limit bars show percentages of CD44high cells.

 
Concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid increases phenotypically activated NK1.1high NK cells and CD8+ T cells

The immunostimulatory action of IL-2 has been tested for clinical use to target several types of cancer and HIV infection (1). One obstacle to the clinical use of IL-2 is its short half-life in the circulation (28, 29). We hypothesized that the ability of the anti-IL-2 mAb to retain IL-2 in vivo would be beneficial for IL-2 immunotherapy, especially when treatment with the mAb was combined with delivery of the IL-2 plasmid DNA. Therefore, we investigated the possibility of using concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid DNA to generate a sustained immune activation.

After the anti-IL-2 mAb plus IL-2 plasmid DNA treatment, a time course analysis revealed that the percentage of CD8+ and NK1.1high (mostly CD4–CD8–) populations in the total PBL substantially increased, reaching ~50% and 30%, respectively, on day 5 (Fig. 6A). The increase in the NK1.1high population declined thereafter, but the CD8+ population remained at >40% of the PBL until at least day 12. Treatment with the IL-2 plasmid DNA alone (plus rat IgG) did not significantly affect the CD8+ population and slightly but significantly increased the NK1.1+ population (Fig. 6A). After the IL-2 plasmid DNA treatment, the activation marker CD69 was clearly up-regulated on the NK1.1high population, and this up-regulation was more prolonged when the anti-IL-2 mAb was coinjected with the plasmid (Fig. 6B, bottom). Interestingly, the anti-IL-2 mAb plus IL-2 plasmid DNA treatment also selectively induced CD44highCD8+ T cells expressing NK1.1 molecules (Fig. 6C). The NK1.1 level on the CD44highCD8+ T cells was lower than on the TCRbeta–NK1.1high cells (data not shown). It is reported that IL-2-activated splenocytes from NKT cell-deficient mice acquire the expression of NK cell-associated molecules including NK1.1 on CD8+ T cells (30). In addition, IL-2-activated CD44highCD8+ T cells with NK receptors can kill syngeneic tumor cells in vitro (25). We examined the existence of anti-IL-2 Ab in serum and observed the Ab existed at least 25 days after the final injection of the Ab (1 mg in 5 i.p. injections) by Western blotting (data not shown). We also showed that the existence of the complex between IL-2-His and anti-IL-2 Ab (around 200 Kd in a nonreducing condition) as well as IL-2-His alone (between 28 Kd and 21 Kd in nonreducing and reducing conditions) in serum at least 3 days after the treatment of a expression vector carrying IL-2-His plus the anti-IL-2 Ab in vivo under nonreducing conditions (Fig. 6D). These results suggest that the combined treatment of the anti-IL-2 mAb plus the IL-2 plasmid induces a prolonged activation state in NK cells and CD8+ T cells in vivo.


Figure 6
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  		FIGURE 6. Concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid phenotypically activates CD8+ T cells and NK1.1high cells. A, Mice were treated with 1 mg of Abs and 6.25 µg of pcDNAs, as indicated. CD8+ (top) and NK1.1high (bottom) populations in the PBL after the injection were examined. Data indicate a percentage in the lymphocyte population. Bar, mean ± SEM (n = 3–4). B, The same mice in A were used to determine the CD69 levels on the NK1.1high population. Treatment groups are indicated as follows: open histograms, rat IgG plus control plasmid DNA; dashed histograms, anti-IL-2 mAb plus control plasmid DNA; solid histograms, rat IgG (top) or the anti-IL-2 mAb (bottom) plus IL-2 plasmid DNA. C, The splenic CD8+ T cell population 5 days after treatment was examined for CD44 and NK1.1 levels. Plots are gated on the TCRbeta+CD8+ population. D, Mice were treated with 1 mg of Abs and 6.25 µg of pcDNA carrying IL-2 fused with His tags (6xHis). Three days after the treatment, serum was collected. Western blotting analysis was performed by anti-His Ab under nonreducing and reducing conditions to detect the complexes carrying the IL-2-His and/or IL-2-His molecules.

 
Concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid enhances Ag-specific CTL activity in vivo

To investigate whether the expansion of activated CD8+ T cells after treatment with the anti-IL-2 mAb plus IL-2 plasmid was functional, Ag-specific CTL activity was evaluated in vivo. Mice that had received an injection of OVA-specific CD8+ T cells (OT-1 T cells) on day –2 were treated with combinations of Abs plus plasmid DNAs on day 0. Equal numbers of target cells, which were pulsed with the OVA257–264 peptide that was recognized by the OT-1 cells and labeled with a high concentration of CFSE, and nontarget cells with low CFSE labeling and without the peptide pulse were injected on day 6 after the treatment. After 12 h, the CFSE levels in the spleen were examined to assess the target-specific CTL activity. Although the IL-2 plasmid alone (plus rat IgG) had some killing effect, the target population was almost completely eliminated by the coinjection of the anti-IL-2 mAb and the IL-2 plasmid (Fig. 7). This result indicates that concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid substantially augmented the Ag-specific CD8+ T cell responses in vivo.


Figure 7
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  		FIGURE 7. Concurrent treatment with the anti-IL-2 mAb and the IL-2 plasmid DNA enhances Ag-specific CTL activity in vivo. Mice that received OT-1 T cells were treated with 1 mg of Abs and 6.25 µg of pcDNA, as indicated. Target cell killing activity was evaluated on day 7, which was 12 h after i.v. injection of a 1:1 mixture of OT-1 peptide-pulsed (CFSEhigh) and nonpulsed (CFSElow) splenocytes from B6.SJL mice. Histograms are gated on CD45.1, which distinguished the CFSE-positive populations from the host cells. The ticked bar shows percentage of CFSEhigh or CFSElow cells.

 
Concurrent treatment with the anti-IL-2 mAb and IL-2 plasmid after tumor injection elicits an effective antitumor immune response

The simultaneous administration of the anti-IL-2 mAb and the IL-2 plasmid generated sustained activation of CD8+ T cells and NK cells. Both of these populations are known to be involved in tumor immunity (24, 25). Therefore, we investigated whether the combined use of the anti-IL-2 mAb and IL-2 plasmid would prevent tumor progression in a murine B16 lung metastasis model. In this experiment, a single simultaneous treatment with the anti-IL-2 mAb and IL-2 plasmid was performed 2 days after the mice were inoculated with B16 melanoma cells by i.v. injection. On day 14 (i.e., 12 days posttreatment), tumor metastasis was evaluated by counting the B16 nodules in the lungs. We found that the combined use of the anti-IL-2 mAb and the IL-2 plasmid was effective and significantly reduced the number of B16 nodules in the lung, whereas the IL-2 plasmid alone (plus rat IgG) only slightly decreased the number of nodules (Fig. 8). These results clearly demonstrated that therapeutic treatment consisting of the concurrent administration of the anti-IL-2 mAb and the IL-2 plasmid effectively protects mice from tumor metastasis in this tumor inoculation model.


Figure 8
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  		FIGURE 8. Antitumor immunity is augmented by the concurrent use of the anti-IL-2 mAb and IL-2 plasmid. Mice were inoculated i.v. with B16 melanoma cells on day 0. On day 2, the tumor-bearing mice were treated with 6.25 µg of pcDNA plasmid DNA and 1 mg of Abs, as indicated. Lung metastasis was assessed on day 14. A, Representative images of the lungs 14 days after the B16 inoculation (i.e., 12 days posttreatment). B, B16 nodules on the lungs were counted on day 14. Each column represents the mean + SEM (n = 3–5). **, p < 0.01 vs treatment with rat IgG and control plasmid DNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated 1) that a neutralizing anti-murine IL-2 mAb (clone S4B6) sustained IL-2 in the circulation in vivo and thereby enhanced its biological functions, and 2) that the combined treatment with the anti-IL-2 mAb and the IL-2 plasmid strongly provoked antitumor immunity in mice. These unexpected functions of the anti-IL-2 mAb became more evident when IL-2 was overexpressed (Figs. 1, 2, and 6–8) or injected as a mAb/rmIL-2 immune complex (Fig. 3). In addition, the anti-IL-2 mAb potentially effects the accumulation of endogenous IL-2 so that it exerts biological activity on CD8+ T cells for extended periods (Fig. 5).

We have investigated T cell homeostasis in vivo and reported that memory CD8+ T cells undergo increased cell divisions in vivo when IL-2 is depleted by the anti-IL-2 mAb (12, 13, 14). The increased division of memory CD8+ T cells after anti-IL-2 mAb treatment is associated with a reduction in CD25+CD4+-regulatory T cells (13), is independent of IL-15, and requires CD122 expression on memory CD8+ T cells (14). From these observations, we concluded that a novel putative CD122-targeted cytokine was probably involved in this process (14). The findings in the present study revealed an IL-2-enhancing activity of the anti-IL-2 mAb and may challenge the conclusion of our previous report. However, our experimental protocol of anti-IL-2 mAb injections that led to increased divisions of memory CD8+ T cells actually reduced the Foxp3+CD4+ and CD25+CD4+-regulatory T cells in vivo (Fig. 4 and Ref. 13). A recent report by Fontenot et al. (4) using foxp3gfp mice demonstrated genetically that IL-2 is critical for the peripheral maintenance of regulatory T cells, suggesting that although an increased proliferation of memory CD8+ T cells was induced by the anti-IL-2 mAb treatment, the in vivo IL-2 level is reduced by this treatment to levels that are insufficient to support the survival of regulatory T cells. In addition, a negative effect of IL-2 on the maintenance of memory CD8+ T cells in IL-2 KO mice is also reported (31). Furthermore, there is experimental evidence that human amniotic fluid lacks IL-2 and IL-15 but can interact with CD122 (32). These observations support the conclusions from our previous studies. However, a more careful re-examination of the in vivo relationship between IL-2 and the maintenance of memory CD8+ T cells is necessary. In any case, we originally were looking for another CD122-binding cytokine to explain early work with this Ab (14), and it is not clear from the results here whether that question was answered or explained entirely with the data presented here. Additional studies to answer this question would be warranted.

Similar to the anti-IL-2 mAb, some other Abs against cytokines are reported to increase the in vivo levels of their target cytokine, thereby enhancing their biological activities in most cases. These include Abs against human IL-2 (16, 17, 18, 19), human IFN-{alpha} (20), and murine IL-3, IL-4, IL-6, and IL-7 (21, 22, 23). The precise mechanisms underlying the agonist action of these Abs are still unclear, but the following possibilities can be proposed. First, the anti-IL-2 mAb could reduce the clearance of IL-2, which is reported to take place in the kidney rather than in the liver in rodents (28, 33). Because small proteins (<50 kDa) are filtered in the kidney (34), the binding of IL-2 molecules to a macromolecule such as IgG would prevent their renal clearance, as reported for polymer-attached IL-2 (35). Second, the mAb could act as a carrier protein of IL-2. The in vivo half-lives of IgGs are dependent on the subtype. Rat IgG2a, the subtype of the anti-IL-2 mAb S4B6, has a half-life of ~10 days (beta phase) in mice (36), which is much longer than that of IL-2 (28, 29, 35). Although we did not precisely determine the half-life of the IL-2/mAb complex, this relatively long half-life of rat IgG2a could contribute to sustaining the IL-2/mAb complex in vivo. The stability of the IgG also explains the long term effect of the mAb on the CD44highCD8+ population (Fig. 5). Consistent with this, we observed the Ab against IL-2 was existed at least 25 days after final injection of the Ab (1 mg in 5 i.p. injections) by Western blotting (data not shown). Because we also showed that the complex between IL-2-His and anti-IL-2 Ab was observed in serum at least 3 days after treatment of a expression vector carrying IL-2-His plus the anti-IL-2 Ab in vivo using Western blotting under nonreducing conditions (Fig. 6D), we hypothesize that there are certain amounts of SDS-stable complex between IL-2 and anti-IL-2 Ab in serum after injection of anti-IL-2 Ab for a certain time period. Because CD44high memory CD8+ T cells have a long life span and show slow homeostatic proliferation (12, 37), it is possible that the anti-IL-2 mAb was present and effective in vivo for at least 50 days, whereas the CD44highCD8+ population increased (Fig. 5). The reported finding that the level of CD25+CD4+-regulatory T cells is reduced for 2 mo after a single i.v. injection of the same anti-IL-2 mAb clone supports our observation of a long term effect of the mAb (2). Third, the mAb could protect IL-2 from physiological inactivation, such as via proteolysis. Finkelman et al. (22) reported that neutralizing mAbs for IL-4 (11B11 and BVD4.1D11.2), but not a nonneutralizing anti-IL-4 mAb (BVD6.24G2.3), increased IL-4 bioactivity in vivo, suggesting that the masking of an important region of the cytokine, such as a receptor contact site, plays a key role in this effect.

T regulatory cell percentage has been recovered within 2 mo, but the memory/activated phenotype of the conventional T cells has not been recovered at this time point after anti-IL-2 Ab treatment (Fig. 5 and data not shown). Thus, we hypothesize that the Ab/IL-2 complexes cannot delete functional T regulatory cells but can stimulate the conventional T cells. It is possible that there might be two modes of effect of the Ab: 1) a soluble Ab efficiently neutralizes IL-2 to reduce the T regulatory cells; and 2) the antibody-IL2 complexes can stimulate CD8 and NK cells but not neutralize extra IL-2 in circumstance, which is critical for T regulatory cell survival. Therefore, we hypothesize that the described effect is the results of both receptor agonism (on CD8 and NK cells) and antagonism (on T regulatory cells) of the Ab. We actually spent much time for the experiments to identify the mechanism of the Ab-mediated proliferation/activation of T cells. These experiments include the stimulation of CD44highCD8, CD4, and CTLL2 cells in the combination of rmIL-2, anti-IL-2, anti-CD3 and/or splenic CD11c+ dendritic cells. However, we have always detected that addition of excess amount of the anti-IL-2 Ab did not enhance but suppressed IL-2-induced survival/proliferation of the T cells tested in vitro (data not shown), although the complex formed in vitro stimulated CTLL2 cells just like IL-2 plus control IgG (data not shown) and induced proliferation of CD8 T cells in vivo (Fig. 3). Our data indicate that formation of IL-2 and anti-IL-2 mAb complex in advance of injection is not necessary for the effect of the Ab to show the effect of the Ab in vivo, because we confirmed this point by using a different route of injections (i.e., conditioned medium containing IL-2 i.p. and anti-IL-2 mAb i.v.; data not shown). In in vitro, the S4B6 is well known to have neutralizing activity to CD4 T cells. We have similar in vitro results using CD8 T cells, CD4 T cells, and CTLL2, although an excess amount of anti-IL-2 mAb relative to IL-2 is required for significant inhibition of IL-2 activity in vitro (data not shown). Because we did not observe the enhancing effect of IL-2 by anti-IL-2 mAb in vitro but observed that the preformed complex between the Ab and IL-2 stimulated CTLL2 cells just like IL-2 plus control IgG (data not shown), we speculate that it is due to metabolic advantages of the IL-2/anti-IL-2 mAb complex in vivo, although we do not completely exclude the other possibilities.

Extensive mutational studies of IL-2 revealed that an intact N-terminal portion (Q26–Y45) is required for its recognition by the anti-IL-2 mAb (9). This region contains an amino acid residue (D34) within {alpha} helix A that is critical for the CD122 binding of IL-2 (10). In addition, the peptide encompassing {alpha} helix A of human IL-2, termed p1–30, has an agonist effect through CD122 (11). Although we do not have direct evidence that the anti-IL-2 mAb blocks the binding of IL-2 to CD122, these lines of evidence suggest that the mAb can mask the important region of IL-2, showing a neutralizing potential at least in vitro (Ref. 9 ; data not shown) and for Foxp3+CD4+ regulatory T cells in vivo (Fig. 4 and Refs. 2 and 13). In addition, in vivo treatment with a protease inhibitor, pepstatin, increases the in vivo half-life of IL-2 (33). Therefore, we speculate that the anti-IL-2 mAb also protects the receptor-contact site of IL-2 in vivo, which may curtail its physiological inactivation. The ELISA-detectable free form of serum IL-2 was greater after the anti-IL-2 mAb plus IL-2 plasmid DNA treatment than after the rat IgG plus IL-2 plasmid treatment (Fig. 1A), suggesting the dissociation of bioactive IL-2 from the IL-2/mAb complex. The on-off cycles between IL-2 and the mAb may contribute to the IL-2 enhancing effects of the mAb.

We noticed that after treatment with the anti-IL-2 mAb plus IL-2 plasmid DNA, CD4+ T cells did not expand as vigorously as did NK cells and CD8+ T cells (Fig. 2B). It is reported that NK cells and CD44highCD8+ T cells express higher levels of CD122 than do CD44highCD4+ T cells (38), and CD8+ T cells proliferate in response to IL-2 more sensitively than do CD4+ T cells (39). Therefore, the selective expansions of NK cells and CD44highCD8+ T cells may simply be due to the difference in sensitivity of these cell populations to IL-2 and competition between them. However, treatment with the IL-2 plasmid alone increased the levels of CD4+ T cells as well as of NK cells and CD8+ T cells, compared with the control plasmid (Fig. 2B). Because the anti-IL-2 mAb and IL-2 plasmid treatment activated CD8+ T cells and NK cells (Fig. 6, B and C), it would be intriguing to learn whether the activated effector cells actively suppress the proliferation and/or survival of CD4+ T cells.

IL-2 has been tested for clinical use targeting various types of cancer and HIV infection, due to its potent immunostimulatory properties (1). Because the half-lives of rIL-2 in human and mouse are on the order of minutes (28, 29, 35), a high dose and frequent injections are required to obtain sufficient therapeutic efficacy. Several strategies have been used to increase the bioavailability of IL-2. These include changing the injection route (40), using polyethylene glycol-IL-2 (41), an IL-2-IgG fusion protein (42), proteolysis-resistant IL-2 (43), an IL-2-albumin fusion protein (44, 45), and gene therapy (46). The present study adds an alternative means to maintain and enhance IL-2 efficacy in vivo by combined treatment with the anti-IL-2 mAb and IL-2 plasmid DNA. One great advantage of this combined treatment is its potent effects with only a single administration. Most interestingly, vigorous and preferential expansion of NK1.1highTCRbeta– classical NK cells and NK1.1+CD44highCD8+ T cells, which are known to have antitumor effects (24, 25, 47), was observed after the combined treatment, which probably explains the efficient prevention of lung metastasis by this therapeutic regimen (Fig. 8). Moreover, critical roles of cooperation between NK cells and CD8 T cells for the rejection of B16 tumor cells in vivo has been reported. Markovic et al. (48) have shown that CD8 T cells regulate IFN-mediated NK cell activity against B16 melanoma cells in vivo. Another study demonstrated that NK cells play a crucial role in generation of the CTL response against B16 melanoma cells (49). Recently, Xu et al. (50) have shown a synergistic interaction between NK cells and CD8 T cells in rejecting B16 melanoma in response to IL-12 treatment. All these studies indicate that interactions between NK cells and CD8 T cells play an important role in the rejection of cancer cells. We here showed that both NK cells and CD8 T cells significantly increased after the combined treatment of anti-IL-2 Ab plus IL-2 expression in vivo. Therefore, it is reasonable that both NK cells and CD8 T cells, which increased after the treatment, are critical for the rejection of B16 tumor cells. Consistent with this, we observed there were clustering of CD3+ and CD8+ T cells as well as CD3– and NK1.1+ NK cells in the nodules of B16 tumors in lung after the treatment of anti-IL-2 Ab plus IL-2 expression (data not shown). Therefore, we hypothesize that the cooperation between CD8 T cells and NK cells that are increased by the combined treatment of IL-2-plasmid plus anti-IL-2 Ab plays a critical role for the rejection.

In summary, we have described unexpected actions of the anti-IL-2 mAb S4B6 that augment IL-2 bioactivities in vivo, especially when IL-2 is abundant. Furthermore, we demonstrated that combined immunotherapy using the anti-IL-2 mAb and plasmid DNA delivery of IL-2 effectively attenuates lung metastasis in mice. Our results suggest the concurrent use of neutralizing anti-IL-2 Ab with gene delivery as an alternative strategy for enhancing and maintaining cytokine functions for therapeutic purposes.


   Acknowledgments
 
We thank Dr. Phillipa Marrack (National Jewish Medical and Research Center) for the gift of the S4B6 and TMbeta1 hybridomas; and Drs. William R. Heath (and Eliza Hall Institute of Medical Research), Shin-ichiro Fujii (RIKEN RCAI), and Jun-ichi Miyazaki (Osaka University Graduate School of Medicine The Walter) for generously providing OT-1 TCR-transgenic mice, B16 melanoma cells, and the pCAGGS vector, respectively. We also thank Dr. Hidehiro Kishimoto (Tokyo University of Science) for productive discussions and Ms. Ryoko Masuda and Ms. Mizuki Shimura for their excellent secretarial assistance and Ms. Yuko Ishikura for experimental assistance.


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


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan and by the Osaka Foundation for the Promotion of Clinical Immunology. Back

2 Address correspondence and reprint requests to Dr. Masaaki Murakami, 2-2 Yamadaoka, Suita, Osaka 565-0871 Japan. E-mail address: murakami{at}molonc.med.osaka-u.ac.jp Back

3 Abbreviations used in this paper: Cy, carbocyanin; rh, recombinant human; rm, recombinant murine; IL-2-His, IL-2 tagged with 6x His at the C terminus; WT, wild type. Back

Received for publication March 2, 2006. Accepted for publication April 20, 2006.


   References
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 Materials and Methods
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