,
* Third Department of Internal Medicine, University of Tokushima School of Medicine, Japan; Departments of
Surgery and
Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh Cancer Institute, Pennsylvania; and
Department of Surgery, Institute of Medical Science, School of Medicine, University of Tokyo, Japan
Correspondence: Yasuhiko Nishioka, Third Department of Internal Medicine, University of Tokushima School of Medicine, Kuramoto-cho 3, Tokushima 770-8503, Japan. E-mail: yasuhiko{at}clin.med.tokushima.u.ac-jp
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(IFN-) resulted in a dose-dependent suppression of MLR in mice. The treatment with NG-monomethyl-L-arginine (L-NMMA) completely abrogated IL-12- and IFN--mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of L-NMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34+ cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.
Key Words: MLR • IFN- • L-NMMA
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(IFN-) production and T helper cell type 1 (Th1) differentiation, and antiangiogenic effects [1
2
3
4
], have been reported. Exogenous IL-12 is effective for infectious and malignant diseases [1
2
3
4
5
6
]. Furthermore, IL-12 gene transduction into tumors strongly suppressed the growth of various tumors in vivo [7
, 8
], whereas the mechanisms involved in the antitumor effects of IL-12 are still unclear as a result of the complexity of the biological effects of IL-12. Conversely, the unexpected effects of IL-12 on the cellular immune responses have been reported in mice. Noguchi et al. [9 ] showed that administration of a low dose of IL-12 (1 ng/mouse) enhanced CTL activity specific for the mutated p53 peptide (234CM) and regressed the MethA tumor expressing it, whereas a high dose of IL-12 (100 ng/mouse) suppressed 234CM-specific CTL generation. Martinotti et al. [8 ] reported that a CD8+ T cell-mediated in vivo-immune response against IL-12-transduced colon cancer cells was observed only when CD4+ T cells were abrogated. Furthermore, Orange et al. [10 ] reported that administration of IL-12 decreased antiviral CTL activity in the lymphocytic choriomeningitis virus (LCMV) infection model. The report presented by Piccotti et al. [11 ] demonstrated that IL-12 antagonism using anti-IL-12 antibodies (Ab) or an IL-12 p40 homodimer exacerbated cardiac allograft rejection in mice. Recently, Kurzawa et al. [12 ] clearly demonstrated the suppressive effects of IL-12 on cellular immune responses using syngeneic and allogeneic murine tumor systems. Further studies reported by Koblish et al. [13 ] clarified that nitric oxide (NO) produced by macrophages was responsible for IL-12-mediated immune suppression in mice in vitro and in vivo. On the basis of these results, they suggested the monitoring of suppressive effects of IL-12 when used for malignant and infectious diseases in humans.
DCs are known to be specialized antigen-presenting cells (APCs), which exist in virtually every tissue, capture antigens in situ, and migrate to lymphoid organs to activate naive T cells and play a key role in the induction of primary immune responses [14
]. As murine DCs are also known to be the producer of NO [15
], we examined whether NO derived from DCs is a suppressive factor for the cellular immune responses in allogeneic mixed leukocyte reaction (MLR) stimulated by IL-12 in mice and humans. It is interesting that a high dose of IL-12 suppressed alloreactive proliferation of T cells in mice but enhanced it in humans. Furthermore, we found the high production of NO by murine DCs, whereas neither human CD34+ progenitor cell- nor monocyte-derived DCs produce a significant amount of NO even after stimulation with IFN- and/or lipopolysaccharide (LPS) as well as MLR. These results suggest that immune responses mediated by IL-12 in mice are, at least in part, different from that in humans and that the suppressive effect of IL-12 on cellular immune responses might not be the case in humans.
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was purchased from Boehringer Mannheim (Indianapolis, IN). Specific activities were >5.0 x 106 U/mg and >2.0 x 107 U/mg, respectively. NG-monomethyl-L-arginine (L-NMMA) and NO-generating agent S-nitroso-N-acetyl-penicillamine (SNAP) were purchased from Sigma Chemical Co. (St. Louis, MO).
Cell lines and mouse strains
EL-4 and P815 were generous gifts of William H. Chambers (University of Pittsburgh, PA). These cell lines were maintained in CM as described previously [7
].
Female 6- to 8-week-old C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). Female 6- to 8-week-old BALB/cj mice were purchased from the Jackson Laboratory (Bar Harbor, ME). These animals were used for all experiments at the age of 8–10 weeks old [7 ].
Culture of mouse bone marrow (BM)-derived DCs
BM–DC culture was obtained using methods described previously [16
17
18
19
]. Briefly, murine BM cells were harvested from the femur and tibia of killed mice. Contaminating erythrocytes were lysed with 0.83 M NH4Cl buffer, and lymphocytes were depleted with a cocktail of antibodies (RA3-3A1/6.1, anti-B220; 2.43, anti-Lyt 2; and GK1.5, anti-L3T4; all from American Type Culture Collection, Manassas, VA) and rabbit complement (Accurate Chemical and Scientific Corp., Westbury, NY) on day 0. These cells were cultured overnight in CM to remove the adherent macrophages, and then nonadherent cells were placed in fresh CM containing rm granulocyte macrophage-colony stimulating factor (GM-CSF; 1000 U/ml) and rmIL-4 (1000 U/ml; Shering-Plough, Kenilsworth, NJ; DC media) on day 1. Cells were generally harvested on day 6. BM–DCs were defined by morphology, phenotype, and strong mixed lymphocyte reaction-stimulating activity. Phenotypic analysis by flow cytometry showed a high expression of CD11b, CD11c, CD80, CD86, as well as major histocompatibility complex class I and class II in the majority of the cultured cells (60–95%) [19
]. These DCs were used for the subsequent experiments as immature DCs.
Flow cytometry
For phenotypic analysis of DCs, phycoerythrin- or fluorescein isothiocyanate-conjugated monoclonal Ab (mAb) against murine and human cell-surface molecules [CD11b, CD11c, CD80, CD86, Gr-1, H-2Kb, I-Ab for murine, CD1a, CD14, CD40, CD54, CD80, CD86, human leukocyte antigen (HLA)-ABC, HLA-DR, and appropriate isotype controls, all from PharMingen, San Diego, CA] were used. DCs were stained and analyzed on a FACScan using LYSIS II software (Becton Dickinson, Moutain View, CA) as described previously [19
].
Purification of human CD34+ progenitor cells from BM
Human BM cells were harvested after informed consent. Mononuclear cells (MNC) were purified from human BM cells using density centrifugation with lymphocyte separation medium (LSM; Litton Bionetics, Kensington, MD). These cells were applied for immunoaffinity column (CellPro, Inc., Bothell, WA) after lysis of erythrocytes to purify CD34+ cells [20
]. Purity of CD34+ cells was more than 85%, as determined by flow cytometry. These CD34+ cells were frozen down in liquid nitrogen until used for DC generation.
Generation of human CD34+ progenitor cell-derived DCs
On day 0, CD34+ cells (5x105 cells) were thawed and cultured in six-well plates with cRPMI 1640 with GM-CSF (500 U/ml), tumor necrosis factor 
(50 ng/ml), stem cell factor (R&D Systems, Minneapolis, MN; 10 ng/ml), and follicular lymphoma (Immunex, Seattle, WA; 50 ng/ml) as described previously [21
, 22
]. Half of the medium was changed, and the cells were split at 1:2 every 3–4 days. On day 14, floating and loosely adherent cells were harvested and used as immature DCs, which were defined by morphology, phenotype, and strong mixed lymphocyte reaction-stimulating activity. These cells were highly positive for classes I and II, CD1a, CD40, CD80, and CD86 (39–91%) and weakly positive for CD83 (15–25%) [23
].
Generation of monocyte-derived DCs
Leukocyte concentrates from healthy donors were harvested after informed consent and separated into peripheral blood MNC (PBMC) by density gradient centrifugation in LSM. These cells were suspended at 1 x 106 cells/ml and cultured in six-well plates for 1 h. After being washed twice, the adherent cells were cultured with GM-CSF (500 U/ml) and IL-4 (250 U/ml) for 7 days to generate monocyte-derived DCs [24
25
26
].
MLR in mice and humans
Splenocytes were harvested from BALB/c mice (H-2d). T cells were purified by T cell enrichment column (R&D Systems) after lysis of contaminating erythrocytes. The purity of T cells was 
90%. T cells (2x105 cells) were cultured in 96-well plates with various numbers of BM–DCs irradiated with 1500 rad from C57BL/6 mice (H-2b). On day 3, 50 µl culture supernatants were collected for measurement of NO and enzyme-linked immunosorbent assay (ELISA) of IFN-. 3H-Thymidine (TdR; 1 µCi; ICN Radiochemicals, Costa Mesa, CA) was pulsed for the final 18 h, the culture was terminated, and the incorporation of 3H-TdR was measured as previously reported [27
].
Human PBMC were purified by density centrifugation with LSM. T cells were purified by a T cell enrichment column (R&D Systems) after lysis of contaminating erythrocytes (purity 90%) [25
]. These T cells (2x105 cells) were cultured in 96-well plates with various numbers of allogeneic CD34+ cell-derived DCs irradiated with 1500 rad for 5 days. 3H-TdR incorporation of the reactive T cells was measured as mouse MLR.
Measurement of NO
Human and mouse DCs were stimulated with IFN- (1000 U/ml) and/or LPS (1 µg/ml) for 48 h. Their supernatants were harvested, and NO2- production was assessed using the Griess method [28
, 29
]. Briefly, aliquots of culture supernatant (50 µl) were incubated with 50 µl Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% H3PO4) at room temperature (RT) for 10 min. The absorbance was measured at 550 nm in an automated plate reader. The concentration was determined with reference to a standard curve of sodium nitrite.
ELISA for mhIFN-
The concentration of IFN- was measured by ELISA (PharMingen) [30
]. The lower limit of detection was 15.6 pg/ml. Briefly, microtiter plates were coated overnight with anti-mouse or human IFN mAb at 4°C. The plates were then washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked with PBS containing 10% FBS at RT for 2 h. After washing, the standards and samples were added in the wells and incubated at RT for 4 h. The wells were then washed and incubated with the biotinylated second Ab at RT for 45 min. Finally, the avidine-peroxidase and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) substrate were added in the well, and the absorbance at 490 nm was determined with an ELISA plate reader (Titertek Multiscan, Meckenheim, Germany).
Generation of alloreactive CTL
Mouse alloreactive CTL were generated as described previously [31
]. Briefly, splenocytes were harvested, and T cells were purified as described above. T cells (2x106 cells) from BALB/cj mice (H-2d) were added in 24-well plates to irradiated (1500 rad) DCs (6.7x104 cells) from C57BL/6 mice (H-2b; T cell:DC ratio=30). After 5 days culture, T cells were harvested, and their lytic activity was assessed using a standard 4 h-51Cr release assay against EL-4 (H-2b). The cytotoxicity of these alloreactive CTLs against P815 (H-2d) was reproducibly less than 5%.
Human alloreactive CTLs were generated from peripheral blood lymphocyte-derived T cells. T cells (2x106 cells) purified as described above were cultured with allogeneic irradiated (1500 rad) DCs (6.7x104 cells) generated from CD34+ cells for 5 days. The cytotoxicity of these stimulated T cells was assessed for the same allogeneic DCs used as stimulators. These stimulated T cells did not show any cytotoxicity against autologous DCs generated from PBL with GM-CSF and IL-4.
The cytotoxicity of alloreactive CTL
The cytotoxicity was determined using a standard 4 h-51Cr release assay as described previously [26
, 27
]. In brief, 106 of each of the target cells was labeled with 100 µCi Na251CrO4 for 1 h. After washing twice, these effector and target cells were plated at an appropriate effector:target ratio in 96-well, round-bottom plates. The supernatant (100 µl) was collected after 4 h of incubation, and the radioactivity was counted with a -counter. The percentage of the specific lysis was calculated using the following formula: % specific lysis = 100 x (experimental release–spontaneous release)/(maximal release–spontaneous release).
Statistical analysis
Statistical analysis was performed using the unpaired two-tailed Student’s t-test. The difference was considered significant when the P value was less than 0.05.
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3000 cells/well) were used for MLR as a stimulator and when IL-12 was added at the initiation of MLR (data not shown). However, when human DCs and T cells were used for MLR, IL-12 rather enhanced alloreactive T cell responses, consistent with previous reports [34
] (Fig. 2B) . As it has been clearly demonstrated that IL-12 is one of the growth factors of T cells [1
, 2
], these suppressive effects of mouse MLR by IL-12 seem to be a result of the indirect effect of IL-12 through other factors induced in MLR. IL-12 is known to stimulate IFN- production in T and NK cells [1
, 2
]. Therefore, we examined the production of IFN- in MLR as parallel experiments. As compared with the different effects of IL-12 on alloreactive T cell proliferation in mice and humans, IL-12 equally stimulated IFN- production in mouse and human MLR (Fig. 3
), although the high number of DCs mediated the slight suppression of IFN- production in mice. These results suggested that the second factor induced by IFN- might be responsible for the suppressive effects of IL-12 on mouse MLR.
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Figure 1. The suppressive effect of IL-12 on allogeneic MLR in mice. T cells (2x105 cells/well) were cultured with the indicated number of irradiated (1500 rad) DCs for 4 days. The rmIL-12 (1 ng/ml) was added at the initiation of culture. 3H-TdR was pulsed for the final 18 h. Data are presented as mean ± SDof triplicate culture.
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Figure 2. Addition of IL-12 inhibits the generation of allogeneic MLR in mice but not in humans. Murine (A) and human (B) T cells (2x105 cells/well) were cultured with the indicated number of irradiated (1500 rad) DCs for 4 days. The various doses of IL-12 were added at the initiation of culture. 3H-TdR was pulsed for the final 18 h. Data in MLR are presented as mean ± SDof triplicate culture. Similar results were obtained in three separate experiments.
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Figure 3. IFN- production in murine and human MLR stimulated by DCs and IL-12. Murine (A) and human (B) T cells (2x105 cells/well) were cultured with the indicated number of irradiated (1500 rad) DCs for 4 days. The various doses of IL-12 were added at the initiation of culture. The supernatant (50 µl) was harvested before the addition of 3H-TdR and assessed for IFN- production with ELISA as described in Materials and Methods. Data are presented as mean ± SD of triplicate culture. Similar results were obtained in three separate experiments.
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suppressed the allogeneic MLR by DCs in mice, and L-NMMA completely reversed the suppressive effects of IFN- on MLR by DCs in mice. It is interesting that the addition of IFN- also suppressed the allogeneic MLR in mice (Fig. 4 A
). These effects were reproducibly observed when more than 10 U/ml IFN- was used. A recent report by Bonham et al. [15
] showed that murine DCs, irrespective of their maturation stage, could produce NO in response to IFN- and/or LPS as well as in the allogeneic MLR. In this report, NO was shown to be the mediator for DC apoptosis. However, previous reports refer to the important role of NO as the inhibitor of T cell proliferation [35
36
37
38
]. To examine whether NO played an important role in IFN--mediated suppression of mouse MLR, we tested the effect of an addition of L-NMMA, an inhibitor of NO synthase (NOS). As shown in Figure 4B , L-NMMA (0.5 mM) completely reversed the suppressive effect of IFN- on MLR.
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Figure 4. Addition of IFN- inhibits the induction of allogeneic MLR in mice, and L-NMMA abrogates the IFN--mediated suppression of MLR. T cells (2x105 cells/well) were cultured with the indicated number of irradiated DCs for 4 days. The various doses of IFN- were added at the initiation of culture (A). L-NMMA (0.5 mM) was also added in MLR in the presence of 1000 U/ml IFN- (B). Data in MLR are presented as mean ± SD of triplicate culture. Similar results were obtained in two separate experiments.
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production, which was induced by the addition of IL-12, was further enhanced by L-NMMA in mouse MLR. In contrast, L-NMMA did not produce any effect on the proliferation and IFN- production in human MLR (Fig. 5)
.
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Figure 5. L-NMMA abrogates the IL-12-mediated suppression of mouse allogeneic MLR, but human allogeneic MLR was not affected by the addition of L-NMMA. T cells (2x105 cells/well) were cultured with the indicated number of irradiated DCs for 4 days. L-NMMA (0.5 mM) was added to the allogeneic MLR of mice and humans. IL-12 (1 ng/ml) was used in these experiments. IFN- production in MLR was also assessed with ELISA. Data in MLR are presented as mean ± SD of triplicate culture. Similar results were obtained in three separate experiments.
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and/or LPS, but human CD34+ cell- and monocyte-derived DCs do not. In this experiment, T cells from neither mice nor humans produced a detectable level of NO (data not shown). These results suggest that NO produced by DCs might play an important role in the immune response in the mouse system, especially in the presence of IL-12.
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Figure 6. NO2- was detected in mouse MLR with DCs but not human MLR, and the addition of IL-12 significantly enhanced NO production in mouse MLR. T cells (2x105 cells/well) were cultured with the irradiated (1500 rad) DCs at the indicated T cell:DC ratio (T/DC) for 4 days. Their supernatant was harvested to measure the NO production in MLR using the Griess method as described in Materials and Methods. Similar results were obtained in two separate experiments.
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Table 1. Murine BM–DCs but Not Human DCs Produce NO in Response to Various Stimulia
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Figure 7. Effect of addition of SNAP (NO donor) on murine and human MLR. T cells (2x105 cells/well) were cultured with irradiated (1500 rad) DCs (1x104 cells/well) for 4 days. SNAP (250 µM) was added at the initiation of culture. 3H-TdR was pulsed for the final 18 h. Data are presented as mean ± SD of triplicate culture. *, P < 0.01 as compared with the group without SNAP; **, P< 0.001 as compared with the group without SNAP.
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Figure 8. IL-12 enhanced alloreactive CTL activity in human, but L-NMMA was required for the enhancement of alloreactive CTL in mouse MLR with IL-12. Mouse T cells (2x106 cells) from BALB/cj mice (H-2d) were added in 24-well plates to irradiated (1500 rad) DCs (6.7x104 cells) from C57BL/6 mice (H-2b; T cell:DC ratio=30) in the presence or absence of IL-12 (1 ng/ml) with or without L-NMMA (0.5 mM). After 5 days culture, T cells were harvested for a 4 h-51Cr release assay against EL-4 (H-2b). Human alloreactive CTL were generated from PBL-derived T cells. T cells (2x106 cells) were cultured with allogeneic irradiated (1500 rad) DCs (6.7x104 cells) generated from CD34+ progenitor cells in the presence or absence of IL-12 (1 ng/ml) for 5 days. The cytotoxicity of these stimulated T cells was assessed for the same allogeneic DCs as the stimulators. Data are presented as mean ± SD of triplicate culture. Similar results were obtained in two separate experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-12 is known to play an important role in promoting Th1 differentiation and cellular immune responses. The numerous studies demonstrated the antitumor effects of IL-12, whereas the detail mechanisms remained unclear as a result of the complex effects of IL-12. The recent report by Andrews et al. [39
] demonstrated that the antitumor effects of adenovirus expressing IL-12 (AdVmIL-12) were mainly mediated by the inhibition of angiogenesis, as intratumoral AdVmIL-12 treatment effectively regressed the established tumor even in immune-deficient mice, including CD4- or CD8-depleted, severe combined immunodeficiency, Beige, and RAG-2-, NKT-, CD28-, and perforin-knockout mice. Furthermore, Boggio et al. [40
] also reported that IL-12 prevented the spontaneous tumor development through antiangiogenic effects. Conversely, Horvath-Arcidiacona et al. [41
] reported the discrepancy between the inhibitory effects of tumor growth and CTL generation in IL-12-treated mice. They found that administration of IL-12 in tumor-bearing mice eventually resulted in the rejection of tumors, but CTL activity was reduced despite the enhancement of serum IFN-. Noguchi et al. [9
] also showed that administration of a low dose of IL-12 (1 ng/mouse) enhanced CTL activity specific for the mutated p53 peptide (234 CM) and regressed the MethA tumor expressing it, whereas a high dose of IL-12 (100 ng/mouse) suppressed 234CM-specific CTL generation. Orange et al. [10
] also reported that the administration of IL-12 decreased antiviral CTL activity in the LCMV infection model. Furthermore, the report presented by Piccotti et al. [11
] demonstrated that IL-12 antagonism using anti-IL-12 Ab or IL-12 p40 homodimer exacerbated cardiac allograft rejection in mice. Koblish et al. [13
] recently elucidated the mechanisms involved in IL-12-mediated suppression on cellular immune responses. They reported that NO produced by adherent cells in spleen was an important factor in the IL-12-mediated immune suppression, as a NOS inhibitor completely abrogated it [13
]; whereas the concentration of NO was not measured. In this study, we confirmed and extended their study using allogeneic MLR stimulated by DCs and demonstrated that murine DCs as well as macrophages were potential producers of NO. Bonham et al. [15
] and Lu et al. [42
] also demonstrated NO production of murine BM–DCs and that L-NMMA enhanced allogeneic MLR when a high number of DCs were used [15
, 42
].
These results presented above support the possibility that IL-12 has the suppressive effects on allogeneic CTL generation by enhancing NO production. It is not surprising that NO suppresses the mitogenic activity of T cells and development of allospecific CTLs, as the previous reports showed evidence that NO produced by macrophages was a suppressive factor of the proliferation of lymphocytes [43 44 45 ]. However, Piccotti et al. [46 ] verified the enhancement of allogeneic MLR in the presence of IL-12. Bloom and Horvath [47 ] reported that IL-12 did not affect allogeneic MLR. These discrepancies between the previous reports and ours might be a result of the differences of stimulator used in MLR. BM–DCs were used in our experiments and splenocytes in the other as a stimulator. Splenocytes contain a DC population, but the percentage of DCs in the spleen is 1.0–1.6% [48 ]. DCs are stronger stimulators in MLR when compared with splenocytes. Therefore, the stimulation by DCs might lead to a higher production of NO and the subsequent suppression of MLR. As DCs but not other APCs are known to play a key role in the induction of primary immune responses [14 ], experiments with DCs seem to be more important to reflect the host immune response. Our data clearly demonstrated that NO is a suppressive factor presumably produced by mouse DCs in MLR. In addition to the data of NO2- production, the blocking experiments with L-NMMA corroborated the inhibitory effect of NO on mouse MLR. Furthermore, the treatment with L-NMMA clearly enhanced allogeneic CTL activity. These data suggest that the combined use of IL-12 and L-NMMA might be effective for treatment of tumors in mice. In fact, Koblish et al. [13 ] reported that inhibition of NOS function using L-NMMA enhanced IL-12-induced delay of SCK tumorigenesis. Furthermore, as Gajewski and co-workers [49 ] reported, the use of a low dose of IL-12 might be better in combination with DC-based immunotherapy.
In the present study, exogenous IFN- also suppressed allogeneic MLR, and the addition of L-NMMA abrogated IFN--mediated suppression. There are many controversial reports in which IFN- enhances or inhibits CTL generation. The recent study using mice with the IFN- gene knocked out showed that allospecific CTL activity of IFN--/- mice was higher than that of IFN-+/+ mice, indicating that the existence of IFN- somehow inhibits the generation of allospecific CTL [50
]. As IFN- stimulates NO production, it is likely that these results might have been affected by the condition of NO production in culture.
Conversely, exogenous human IL-12 has been reproducibly reported to enhance antigen-specific human CTL induction by DCs from CD8+ T cells in vitro [34
, 39
]. Chouaib et al. [34
] showed that IL-12 was involved in human allogeneic MLR and the addition of rhIL-12-enhanced, allospecific CTL activity using a blocking experiment with anti-human IL-12 antibody. Consistent with these results, we demonstrated in this report that IL-12 (1 ng/ml) enhanced allospecific CTL activity in humans. This difference between murine and human CTL induction by IL-12 was a result of the different production of NO by DCs. The present study was the first report describing that human CD34+ cell- and monocyte-derived DCs did not produce a significant amount of NO in response to LPS and/or IFN- as well as in allogeneic MLR.
These results suggest that NO production by APCs including macrophages and DCs is an important factor regulating the effect of IL-12 in mice. The combined use of L-NMMA might reverse the suppressive effects of IL-12 on the induction of CTLs in mice and more closely mimic immune responses mediated by IL-12 in humans. As NO induced by IL-12 in vivo is likely to be less in humans, the antitumor effect mediated by IL-12 via cellular immune responses might be expected in humans rather than in mice.
Received April 25, 2002; revised December 24, 2002; accepted January 23, 2003.
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production J. Immunol. 153,1697-1706[Abstract] induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect J. Exp. Med. 188,1603-1610 J. Immunol. 160,1139-1147-inducing factor/IL-18 administration mediates IFN-- and IL-12-independent antitumor effects J. Immunol. 160,1742-1749 in the induction of the nitric oxide-synthesizing pathway J. Immunol. 147,144-148[Abstract] Int. Immunol. 8,661-673, endotoxin, and interaction with allogeneic T cells. Nitric oxide production is associated with dendritic cells apoptosis J. Immunol. 157,3577-3586[Abstract] in the induction of the nitric oxide-synthesizing pathway J. Immunol. 147,144-148 genes Science 259,1739-1742This article has been cited by other articles:
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