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The Journal of Immunology, 2007, 179, 4231 -4238
Copyright © 2007 by The American Association of Immunologists, Inc.
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Postimmunization with IFN-{gamma}-Secreting Glioma Cells Combined with the Inducible Nitric Oxide Synthase Inhibitor Mercaptoethylguanidine Prolongs Survival of Rats with Intracerebral Tumors1

Wiaam Badn2, Edward Visse, Anna Darabi, Karin Enell Smith, Leif G. Salford and Peter Siesjö

Glioma Immunotherapy Group, The Rausing Laboratory, Division of Neurosurgery, Department of Clinical Sciences, University of Lund, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
High-grade gliomas are one of the most aggressive human tumors with <1% of patients surviving 5 years after surgery. Immunotherapy could offer a possibility to eradicate remnant tumor cells after conventional therapy. Experimental immunotherapy can induce partial cure of established intracerebral tumors in several rodent models. One reason for the limited therapeutic effects could be immunosuppression induced by both the growing tumor and the induced immune reaction. NO has been implicated in tumor-derived immune suppression in tumor-bearing hosts, and unspecific inhibitors of NO synthase have been shown to boost antitumor immunity. In this study, we show that the inducible NO synthase (iNOS)-specific inhibitor mercaptoethylguanidine (MEG) superiorly enhanced lymphocyte reactivity after polyclonal stimulation compared with the iNOS-specific inhibitor L-NIL and the unspecific NO synthase inhibitor L-NAME. Both iNOS inhibitors increased the number and proliferation of T cells but not of B cells. When combined during postimmunization with IFN-{gamma}-secreting N32 rat glioma cells of rats harboring intracerebral tumors, only MEG increased the cure rate. However, this was only achieved when MEG was administered after immunizations. These findings implicate that NO has both enhancing and suppressive effects after active immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The prognosis for patients with high-grade gliomas is dismal. Even with macroscopic eradication of tumors after surgery, chemotherapy, and radiotherapy, local or distant recurrence arises from tumor cells residing within the normal brain. Immunotherapy could offer a possibility to kill remnant tumor cells after conventional therapy. Postimmunization of tumor-bearing animals can induce cure or prolonged survival in different tumor models, including brain tumors (1, 2). However, the effect of immunotherapy is often partial. One explanation to this could be that immunosuppressive mechanisms develop and down-regulate cytolytic responses. Immune activation often induces immune suppressive mechanisms to avoid over-activation and possible damage of normal tissues. Immunosuppression can be induced directly from tumor cells by secretion of immunosuppressive factors, or indirectly by induction of immune cells or stroma cells with suppressive capacity (3).

Gliomas have been shown to spontaneously produce factors that suppress CTL-mediated antitumor responses, e.g., TGF-beta, IL-10, and PGE2 (4, 5, 6, 7). Recent studies demonstrate that myeloid suppressor cells and tumor-associated macrophages induce immunosuppression via modulation of L-arginine metabolism (8, 9), either via up-regulation of arginase (10, 11), thereby diminishing arginine locally, or via transformation of arginine into NO by inducible NO synthase (iNOS)3 (12, 13). NO or peroxynitrite, a product from the reaction between NO and superoxide, suppresses T cells by impairing IL-2R signaling pathways (14). However, it has been shown that small amounts of NO are essential for Th1 induction (15). Recently it has been shown that NO is synthesized by endothelial NO synthase in T cells and is essential for their activation (16). Furthermore, NO can directly kill tumor cells and thus contribute to the immune response against tumor cells (17, 18). Thus NO could have both an enhancing and a suppressive effect on immune reactivity against tumors.

Previously, we have shown that intracerebral (i.c.) tumors in rats can be eradicated by immunizations with IFN-{gamma}-secreting tumor cells (1). In this tumor model, immunohistochemical analysis of infiltrating cells at the immunization and tumor site showed a specific expression of iNOS in infiltrating macrophages (19). Our previous results show that the nonselective inhibitor L-NAME (N-nitro-L-arginine methyl ester) can be used in vitro to counteract the NO-dependent immunosuppression exerted by adherent splenocytes from tumor-bearing rats with either s.c. gliomas or colon carcinoma (20, 21). Inhibition of NOS using L-NAME has been reported to boost immunotherapy of different experimental tumors (22, 23). However, the selective iNOS inhibitor L-NIL (L-N6-(1-imminoethyl)-lysine) was more effective in enhancing lymphocyte activation than L-NAME and could boost an IFN-{gamma} based immunotherapy in rats with i.c. tumors (24).

Mercaptoethylguanidine(MEG) and L-NIL are both selective inhibitors of iNOS as well as peroxynitrite scavengers (25, 26, 27). Both substances are irreversible inhibitors. The inhibition by MEG is developed from an initially noncovalent complex with the enzyme, to a tight binding with covalent changes to the enzyme or the inhibitor or both (28, 29). In the case of L-NIL, the inhibition involves reaction with the heme group of the enzyme complex, resulting in loss of heme, or forming an unstable heme adduct that breaks down and inactivates iNOS (28). In the present study, comparative experiments were conducted to investigate the effects of MEG and L-NIL in i.c. tumor-bearing rats postimmunized with IFN-{gamma}-secreting tumor cells.


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

Fischer 344 rats were obtained from Scanbur and kept in a temperature-controlled environment. All animal procedures were in agreement with the rules of the Swedish Board of Animal Research and approved by the local board.

Tumor cells and cell culture

The rat glioma cell line N32 was induced by ethyl-N-nitrosourea in vivo (30). The generation of N32 transfectants expressing rat IFN-{gamma} (N32-IFN-{gamma}) has been previously described (1). The cells were cultured in vitro in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Biochrom), 10 mM HEPES, and 0.5 mM sodium pyruvate (Invitrogen Life Technologies).

Immunization

Irradiated (80 Gy from a 37Cs source) N32 or N32-IFN-{gamma} cells, suspended in serum-free RPMI 1640, were used for immunizations. A stable one-cell clone, producing 30.7 ± 0.91 pg IFN-{gamma}/1 x 105 cells/48 h, was used. Rats were immunized i.p. with 3 x 106 cells in 0.2 ml. To study in vivo effects on splenocytes, rats were i.p. immunized once 1 wk before spleen isolation. For survival experiments animals were i.p. immunized three times with 3 x 106 cells at days 1, 14, and 28 after tumor inoculation. For mechanistic experiments rats were immunized twice i.p. at days 1 and 14 after tumor inoculation.

Tumor inoculation

Eight- to 10-wk-old rats were i.c. inoculated with N32 tumor cells (3000 cells/5 µl of R0) in the right nucleus caudatus using a small stereotactic frame (Kopf Instruments). Coordinates were 2 mm to the right and in front of the bregma, and 5-mm deep measured from the outer surface of the skull. The animals were sedated with 2.9% isoflurane. The cells were injected slowly during 2 min to diminish backflow through the insertion canal. The needle was withdrawn after a 2-min delay, and the hole in the skull was sealed with bone wax. Rats were either immunized with N32 or N32-IFN-{gamma} tumor cells, as described. Animals with symptoms of tumor progression were sacrificed and checked for i.c. tumors.

Spleen and lymph node cell preparation

Splenocytes were suspended as previously described (31), briefly by scraping the spleens with a sterile needle in a small amount of RPMI 1640, and were further suspended in 10 ml of RPMI 1640 medium supplemented with FCS (10%) plus 2-ME, as previously described (21, 23). Lymph node cells were prepared by meshing the lymph nodes in a cell strainer, and were taken up in a small volume of 3-ml RPMI 1640 medium with FCS (10%) without 2-ME. Adherent cells were removed by incubation for 45 min at 37°C in a humidified, 5% CO2 atmosphere, in plastic culture flasks. Splenocytes and nonadherent splenocytes were suspended in RPMI 1640 medium supplemented with FCS plus 50 µM 2-ME. The deep cervical lymph nodes (DCLN) were isolated from the tumor-bearing rats and were suspended in RPMI 1640 medium with FCS without 2-ME.

Proliferation assays

Splenocytes (3 x 105 cells/well) were first stimulated in 96-well plates with 0.5 ng/ml staphylococcal enterotoxin A (SEA; BD Pharmingen) for 4 days. For analysis of proliferation, [3H]thymidine (12.5 µCi/well) (Amersham Biosciences) was added 6 h before the end of culture, and the radioactivity was determined in a scintillation counter (Wallac Microbeta). All tests were conducted in six parallel wells. Data are presented as cpm.

For analysis of proliferating T cell subsets, BrdU (2 µg/ml) was added to splenocyte cultures 6 h before FACS analysis. In brief, after Fc receptor blockage, cells were incubated with primary Abs (CD3 and CD45-RA) at 4°C for 30 min in the dark. Cells were fixed with ethanol/paraformaldehyde, stained with anti-BrdU FITC (BD Biosciences), and were analyzed by FACSCalibur flow cytometer (BD Biosciences).

Drugs and administration

NOS inhibitors used were L-NAME (Sigma-Aldrich), L-NIL (Larodan Fine Chemicals), and MEG, a gift from Inotek Pharmaceuticals. For in vivo administration of L-NIL (1 mg/day), mini-osmotic pumps (model 2001; Alzet) were used. L-NIL was dissolved in 0.9% sodium chloride solution and pumps were filled according to the manufacturer’s instructions and i.p. placed. MEG (10 mg/kg four times a day) was injected i.p. due to the inability to use mini-osmotic pumps. The NO donor SNAP (S-nitroso-N-acetylpenicillamine; Sigma-Aldrich) was investigated on tumor cell proliferation.

Cytokine production assay

For cytokine production, supernatants from the spleen cell cultures as described were collected at the end of the culture period and stored at –20°C. The amounts of IFN-{gamma} and IL-10, respectively, were measured from duplicate samples by ELISA kit (OptEIA Set for rat IFN-{gamma} and IL-10; BD Pharmingen).

Measurement of nitrite production

The same supernatants as tested for IFN-{gamma} production were also tested for NO production. This production was measured from duplicate samples as nitrite (NOFormula 2) concentration by the Griess assay (32), where 100 µl of Griess reagent (a 1/1 mixture of 1% p-aminobenzene-sulfonamide in 5% H3PO4 and 0.1% naphtylethylenediamine dihydrochloride in distilled H2O) were added to culture supernatants and standard (NaNO2) in 96-well plates. Plates were incubated at room temperature for 10 min, and absorbance was measured at 550 nm.

Flow cytometry

The expression of CD3, CD4 and CD8, CD25, and FOXP3 in spleen and cervical lymph nodes was analyzed by flow cytometry. The following Abs and fluorochrome-conjugated reagents used were: PE-CD3 (1F4), FITC-CD4, PE-CD4, allophycocyanin-CD4 (OX-35), PE-CD25 (OX-39), and FITC-CD8b (clone 341) all obtained from BD Pharmingen. Allophycocyanin-Foxp3 (FJK-16s) was used together with a permeabilization/fixation kit (eBioscience).

Statistics

The Kruskal-Wallis nonparametric ANOVA was used for evaluation of differences among three or more groups and Mann-Whitney U test for differences between two groups. Log-rank test was used for evaluation of significance in survival data. Two-tailed values of p < 0.05 were considered statistically significant. We performed statistical evaluation using the StatView software (Abacus).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Splenocyte proliferation and IFN-{gamma} production is significantly enhanced after iNOS inhibition in vitro

To study the effect of the different NOS inhibitors during lymphocyte activation, splenocytes were stimulated in vitro with SEA (0.5 ng/ml) and incubated with increasing doses of MEG, L-NIL, or L-NAME for 4 days. Both the specific iNOS inhibitors L-NIL and MEG were potent in enhancing splenocyte proliferation at much lower doses compared with the nonspecific inhibitor L-NAME, which was effective only at one dose (1 mg/ml) (Fig. 1A). L-NAME at lower doses as 1 µg/ml was not at all able to enhance the proliferation of splenocytes (33). Furthermore, L-NIL had a broader therapeutic window than both L-NAME and MEG. To address T cell activation, we also studied cytokine production from splenocytes. IFN-{gamma} production was significantly higher (p < 0.05) than MEG-treated splenocytes already at a dose of 1 µg/ml compared with both L-NIL and L-NAME (Fig. 1B). IL-10 production was also increased after NOS inhibition (Fig. 1C). However, there were no significant differences between the NOS inhibitors concerning IL-10 production. MEG was significantly more effective than L-NIL and L-NAME in inhibiting the production of NO, as the nitrite concentration decreased from 28 to 2.8 µM after treatment with 1 µg/ml MEG compared with a decrease from 27 to 11 µM with L-NIL (Fig. 1D). L-NAME was only able to inhibit NO production at the dose of 1–5 mg/ml, whereas at higher doses it was toxic resulting in cell death (33).


Figure 1
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  		FIGURE 1. In vitro comparison of NOS inhibitors on immune cell activity. Splenocytes were stimulated with 0.5 ng/ml SEA, and were cultured for 4 days. A, Proliferation of the splenocytes after treatment with different doses (µg/ml) of NOS inhibitors MEG, L-NIL, and L-NAME. Mean cpm x 10–3 ± SD are shown (n = 6 parallel wells). Supernatant from these cultures was analyzed for IFN-{gamma} production (ng/ml) (B), IL-10 (ng/ml) (C), and nitrite production (µM) (D). The proliferation (E) and the NO production (F) from total and nonadherent splenocyte populations were compared before and after treatment with different doses of MEG.

 
Due to the fact that the adherent spleen cell population is enriched with cells producing suppressive molecules such as NO and PGE2 (21, 23, 34), we chose to test the effect of MEG after stimulation of either total or nonadherent splenocytes. SEA-stimulated total or nonadherent splenocytes were incubated with increasing doses of MEG for 4 days. Although 1–2 µg/ml MEG was needed to significantly enhance splenocyte proliferation from 6 to 200 x 10–3 cpm, a dose as low as 0.1 µg/ml MEG was able to increase the proliferation of the nonadherent splenocytes to almost the same level, from 20 to 170 x 10–3 cpm (Fig. 1E). There was an inverse correlation (K = –0.43) between proliferation and reduction of NO in the cell culture supernatant, measured as nitrite concentration (Fig. 1F).

iNOS inhibition significantly enhanced the proliferation of T cells in vitro

To assess whether iNOS inhibition influences proliferation of different lymphocyte subsets in the spleen, BrdU flow cytometry was used. Splenocytes were restimulated with SEA and cultured with medium, with 1 µg/ml MEG, or with 1 mg/ml L-NIL for 4 days. Both MEG and L-NIL significantly enhanced (p < 0.05) the proliferation of CD3+ T cells, but not of the CD45RA+ B cells (Fig. 2).


Figure 2
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  		FIGURE 2. Specific iNOS inhibitors induce T cell proliferation from splenocytes. Splenocytes were stimulated with 0.5 ng/ml SEA, and were cultured for 4 days. The percentage of CD3+ and CD45RA+ BrdU+ double-positive splenocytes cultured in medium only, in 1 µg/ml MEG, or with 1 mg/ml L-NIL is shown. One representative experiment of four with similar results is indicated. Significance was evaluated using Kruskal-Wallis and Mann-Whitney U test.

 
Neither L-NIL nor MEG directly inhibited the growth of the glioma tumor cell lines N32 and N32-IFN-{gamma}

To exclude the possibility that the iNOS inhibitors could directly affect tumor cell growth, we investigated the effect of these inhibitors on the glioma cell lines used in this model. Neither MEG nor L-NIL did affect the proliferation of these tumor cell lines (Fig. 3). Additionally to test the direct effects of NO on tumor cell growth, the NO donor SNAP was used. The proliferation of both cell types was significantly reduced by SNAP (Fig. 3).


Figure 3
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  		FIGURE 3. The effect of MEG/L-NIL and NO on the glioma cell lines in vitro. Proliferation of tumor cells (15,000/well in 96-well plate) N32 ({square}) and N32-IFN-{gamma} ({blacksquare}) cultured in medium only or with the NO donor SNAP (0.3 and 1 mM), with the iNOS inhibitor MEG (1 µg/ml), or with L-NIL (1 mg/ml) for 4 days. Mean cpm x 10–3 ± SD of five replicates are indicated.

 
Combined MEG treatment and postimmunization of glioma bearing rats increased cure rate 3-fold only when MEG was administered after immunizations

We have previously shown that L-NIL could boost an IFN-{gamma} based immunotherapy of rats with i.c. tumors (33). Based on these results we initially combined immunizations with N32-IFN-{gamma} and MEG treatment (10 mg/kg, four times a day) during days 1–7, which is during and shortly after the first immunization. However, surprisingly this schedule significantly decreased (p < 0.05) the survival when compared with animals that received immunizations only (Fig. 4A). We therefore hypothesized that NO is essential during immunization, hence NOS inhibition should be administered in between immunizations. However, MEG treatment during days 7–14, or days 17–24 was not able to prolong the survival (Fig. 4A). Although MEG treatment during or after the first immunization, i.e., days 1–7 or days 7–14, resulted in lesser survival, treatment with MEG did not diminish the survival when administered during days 17–24. We therefore hypothesized that the effect could be increased by iNOS inhibition after more than one immunization. Indeed, the combination of MEG after both first and second immunization (i.e., during days 7–14 and days 17–24) significantly prolonged (p < 0.0005) the survival and increased the cure rate 3-fold of rats with i.c. tumors (Fig. 4B). This effect seemed to be synergistic and was dependent on immunization with N32-IFN-{gamma} cells because replacement with N32 wild-type tumor cell immunization did not prolong the survival but significantly (p < 0.05, log-rank test) shortened it (Fig. 4B). This effect was also related to a significantly decreased (p < 0.05) NO concentration in serum after MEG treatment (Fig. 4C).


Figure 4
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  		FIGURE 4. Survival was significantly prolonged after combination of MEG treatment and immunization with N32-IFN-{gamma} cells. Eight-wk-old rats were i.c. inoculated with N32 tumor cells at day 0. At day 1, 14, and 28, the rats were immunized i.p. with 3 x 106 N32-IFN-{gamma} cells. A, Kinetics of MEG treatment and immunization with IFN-{gamma} glioma cells. Rats were immunized and receiving PBS only, immunized with N32-IFN-{gamma} cells (IMM IFN) or MEG (10 mg/kg every 6 h) during days 1–7, or immunized and treated with MEG during days 7–14 or days 17–24. Data are pooled from two to three experiments. *, p < 0.05 using log-rank test. B, MEG treatment after first and second immunization significantly prolonged survival 3-fold. Rats were either immunized with N32-IFN-{gamma} cells (IMM IFN) or immunized and treated with MEG during both days 7–14 and days 17–24. As a control, rats were immunized with 3 x 106 N32 wild-type glioma cells or were immunized with wild-type glioma cells and treated with MEG during days 7–14 and days 17–24. Data are pooled from one to four experiments. ***, p < 0.0001 using log-rank test. C, The effect of MEG on serum NO concentration was investigated. Tumor-free (TF) or tumor-bearing (TB) rats or tumor-bearing rats immunized with N32-IFN-{gamma} cells were compared with tumor-bearing rats immunized with N32-IFN-{gamma} cells that are also treated with MEG. Data represent mean ± SD of NaNO2 of four to six rats. *, p < 0.05 using Kruskal-Wallis and Mann-Whitney U test. D, In a similar way, the effect of L-NIL on the survival was investigated. Rats were immunized with N32-IFN-{gamma} cells (IMM IFN) and received PBS only ({blacksquare}) or immunized and treated with L-NIL (1 mg/day) during days 7–14, or immunized and treated with L-NIL during days 7–14 plus days 17–24. As a control, rats immunized with N32 wild-type cells (3 x 106) and treated with L-NIL during days 7–14 plus 17–24, and were compared with survival of rats only immunized with N32-wild-type glioma cells (•).

 
Analogous to the results with MEG, combination of L-NIL with the IFN-{gamma}-based immunization during days 7–14 gave less survival than immunization alone (Fig. 4D). Although combination of L-NIL during days 7–14 and days 17–24 did not significantly prolong survival, there was a trend toward it. L-NIL combined with wild-type tumor cells also did not increase survival (Fig. 4D).

Combined MEG treatment and immunization significantly increased T cell numbers in the DCLN of rats with i.c. tumor

In this experiment we aimed to explain the mechanism underlying the prolonged survival after MEG treatment. Therefore, rats with i.c. tumors, immunized days 1 and 14, were treated with MEG during days 7–12 and days 17–22. The tumor, the DCLN, and the spleen were isolated on days 22 and 23. The number of lymphocytes both in DCLN and spleen was significantly increased in the MEG-treated animals compared with controls (Fig. 5, A and B). There was an increased infiltration of CD8 cells in the tumor, and both iNOS and nitrotyrosine were detected with immunohistochemistry. However, there were no differences between control animals and MEG-treated animals (data not shown). Further analysis of the DCLN showed a significant increase in CD3+ T cells and in CD3+CD4+ T cells after MEG treatment, but no change in the CD3+CD4– (CD8+) cell population (Fig. 5C), which concurs with the immunohistochemical staining of CD8 cells at the tumor site (data not shown). However, these changes were not observed in the spleen (data not shown). To exclude the possibility that the effect of MEG treatment depends on changes in the number of regulatory T cells, the CD4+CD25+Foxp3+ cell population in the DCLN and the spleen was studied. MEG did not affect the CD4+CD25+Foxp3+ population in the DCLN (Fig. 5C) or in the spleen (data not shown). However, MEG treatment significantly diminished the number of CD8+CD25+ cells (Fig. 5D).


Figure 5
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  		FIGURE 5. MEG enhances T cell proliferation in vivo. Rats with i.c. glioma were immunized days 1 and 14 and were simultaneously receiving MEG during days 7–12 and days 17–22. Animals were sacrificed on days 22 or 23. The number of lymphocytes (106) in the DCLN (A) and the spleen (B) was compared between MEG-treated and immunized only animals. The mean cell number ± SD of four animals are indicated. C, The cells isolated from the DCLN were stained for T cell markers anti-CD3+ and anti-CD4+ and analyzed by FACS. These populations were increased after MEG treatment (right) compared with control treatment (left). D, The CD4CD25Foxp3+ cell populations in the DCLN were also investigated. However, this population remained unchanged after MEG treatment. The percentage of positive populations is indicated. Data from one of four experiments with similar results are shown. E, The CD8+CD25+ cell populations within the DCLN were investigated after MEG treatment. One representative experiment of five with similar results is indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The aim of this study was to compare the effect of different NOS inhibitors on the immune response when combined with an IFN-{gamma}-based immunotherapy. We show that after polyclonal stimulation, both the iNOS selective inhibitor L-NIL and the nonselective NOS inhibitor L-NAME could enhance proliferation and IFN-{gamma} production of splenocytes, but MEG induced the highest IFN-{gamma} production. Although there was a correlation between the proliferation and nitrite reduction, we cannot exclude other mechanisms mediated by MEG such as inhibition of cyclooxygenase-2 (35). The fact that L-NAME is a nonspecific inhibitor of NOS probably explains the lesser effect and that higher doses were needed to achieve maximal effect. Both MEG and L-NIL have been shown to scavenge peroxynitrite, which could explain their effect vs L-NAME, but not the superior effect of MEG vs L-NIL.

All three inhibitors increased the production of IL-10 during activation, but to a much lesser extent than IFN-{gamma}. The increase in IL-10 levels was not caused by elevated IFN-{gamma} levels, as inhibition of IFN-{gamma} did not affect the IL-10 levels (data not shown). As there were no major differences in IL-10 levels between the NOS inhibitors, we consider that the minor IL-10 increase is not related to the differential effects on lymphocyte activation.

Next we show that the major suppression and NO production originates from the adherent splenocyte population as reported before (21). This was indicated by a lower concentration of NO in the nonadherent population than in the total splenocyte population. NO in the nonadherent population could be produced from the remaining macrophages or dendritic cells that were not removed by adherence, or produced from the activated T cells themselves by induction of endothelial NO synthase (16). The effects observed on nonadherent splenocytes could also be due to a decrease in peroxynitrite and PGE2, which can be inhibited by MEG. Furthermore, the observation that simultaneous inhibition of NO and PGE2 was synergistic (W. Badn, M. Esbjörnsson, L. G. Salford, P. Siesjö, and E. Visse, manuscript in preparation) can explain how low concentration of MEG is sufficient to enhance the proliferation ~10 times. We also show that both MEG and L-NIL significantly support T cell proliferation, and that none of the iNOS inhibitors induced a clear B cell proliferation. However, as the assay used specifically activates T cells, we cannot exclude an effect on B cell under different stimulation conditions.

We could also exclude any effects of NOS inhibitors on the tumor cells used in this model, as proliferation of either N32 or N32-IFN-{gamma} was not altered by MEG or L-NIL (Fig. 3). However, both cell lines were sensitive for NO as demonstrated by a decrease of proliferation after addition of the NO donor SNAP. The cytotoxic effect of NO on tumor cells might contribute to the effect of immunizations, and different studies have shown sensitivity of tumor cells for elevated amounts of NO (17, 36). However, high levels of NO seemed to be favorable only locally at the tumor site, and high systemic levels were shown to suppress the immune response of the tumor-bearing hosts (20, 21, 37). Thus it might be favorable to deliver NO donors intratumorally and combine it with NOS inhibition, at different time points.

Based on these results we show that the combination of a specific iNOS inhibitor MEG with immunization using cells producing IFN-{gamma} resulted in a 3-fold increase in the cure rate of rats with i.c. tumors. Interestingly, this combination did not work unless MEG was administered after both first and second immunization (during days 7–14 and days 17–24). MEG administration during the first immunization, i.e., during days 1–7, even decreased the survival when compared with immunization only. NO release during or shortly after immunization may be due to IFN-{gamma} secreted at the immunization site because we have observed a specific induction of iNOS here after immunization with IFN-{gamma}-transfected cells (19). A possible explanation for this decrease in survival might be that NO at this early stage, is needed both for T cell activation (15) and further degradation of tumor cells. Although we are not aware of any reports showing that MEG can influence the induction of endothelial NO synthase in T cells, we cannot rule out this possibility.

Neither L-NIL nor MEG treatment during day 7–14 prolonged the survival, but when combined with treatment during days 17–24, only MEG significantly prolonged the survival, indicating that it is necessary to at least inhibit NO after the first and second immunization to be able to boost antitumor T cell responses. Another explanation for the need of NO inhibition after second immunization might be that, when the tumor increases in size, more inflammatory cytokines are produced and might induce a higher NO production, which inhibits T cell activation. It could also be that effector T cells obtained after second immunization produce more IFN-{gamma} and other cytokines that induce a high production of NO from myeloid suppressor cells or tumor-associated macrophages. Therefore, later inhibition of NO is needed. Although we could show that the effect of increased cure rate was related to a decrease in NO concentration in serum of MEG-treated animals, the survival outcome from the combination of IFN-{gamma}-based immunization with L-NIL compared with MEG suggests that other suppressive mechanisms could be of importance in this model. Although MEG significantly prolonged the survival, L-NIL was not able to do that. The expression of cyclooxygenase-2 as well as production of peroxynitrite has been observed in many tumor models. MEG has been shown to inhibit both, which might be one of the explanations why MEG is more potent than L-NIL. Another explanation could be that MEG is simply more effective in inhibiting iNOS compared with L-NIL. Both inhibitors affect the cells of the immune system because we have not seen any direct effect on the proliferation of N32 or N32-IFN-{gamma} cells after MEG or L-NIL treatment. Neither of substances per se could influence survival of animals with i.c. tumors. Interestingly immunized tumor bearers had higher NO levels than nonimmunized tumor bearers, indicating that immune activation after immunizations indeed induces NO production.

Increased cell numbers were observed both in the spleen and in the DCLN of MEG-treated animals (Fig. 5, A and B). In addition to that, a significant increase was observed in the CD3+ and CD4+ T cell populations of the DCLN but not spleen after MEG treatment. As the brain parenchyma has been shown to drain to the DCLN, this could signify that T cell homing to the tumor site is increased after MEG treatment or that T cell survival is increased (38). Although several reports have described a role for NO in the effector function of regulatory T cells (37), treatment with MEG did not alter the amount of the CD4+CD25+Foxp3+ regulatory T cells, neither in the DCLN nor in the spleen. Although MEG treatment did not affect the CD4+CD25+Foxp3+ population, we cannot exclude an effect on other populations with suppressive capacity. In this context we observed a reduction of the CD8+CD25+, a population reported to include autoreactive T cells capable of inhibiting CD4+ T cells (39, 40).

In conclusion, this study is the first to show that inhibition of NO using MEG can enhance an IFN-{gamma}-based immunotherapy against experimental i.c. tumors, resulting in a 3-fold higher cure rate. This effect was dependent on administration of MEG after both first and second immunizations, but was quenched when given during immunization. These results indicate the importance of blocking immunosuppressive mechanisms in active immunotherapy of tumors.


   Acknowledgments
 
We thank Catarina Blennow and Suzanne Strömblad for excellent technical assistance. W. Badn designed research, performed research, analyzed data, and wrote the paper. E. Visse designed research, performed research, and wrote the paper. A. Darabi analyzed data. K.E. Smith assisted in writing the paper. L.G. Salford designed research and P. Siesjo supervised, designed research, analyzed data, and wrote the paper.


   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 grants from The Child Cancer Foundation, The Jonas Foundation, The Hedvig Foundation, The Skåne Regional Funds, and the Gunnar Nilsson Cancer Foundation. Support was also from the Swedish Cancer Foundation and the Hans and Märit Rausing Charitable Foundation. Back

2 Address correspondence and reprint requests to Dr. Wiaam Badn, Division of Neurosurgery, Department of Clinical Sciences, BMC:I12, University of Lund, SE-221 84 Lund, Sweden. E-mail address: wiaam.badn{at}med.lu.se Back

3 Abbreviations used in this paper: MEG, mercaptoethylguanidine; iNOS, inducible NO synthase; i.c., intracerebral; SEA, staphylococcal enterotoxin A; DCLN, deep cervical lymph node. Back

Received for publication June 22, 2007. Accepted for publication July 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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